Polyspecific immunoconjugates and antibody composites for targeting the multidrug resistant phenotype

ABSTRACT

Polyspecific immunoconjugates and antibody composites that bind a multidrug transporter protein and an antigen associated with a tumor or infectious agent are used to overcome the multidrug resistant phenotype. These immunoconjugates and composites also can be used diagnostically to determine whether the failure of traditional chemotherapy is due to the presence of multidrug resistant tumor cells, multidrug resistant HIV-infected cells or multidrug resistant infectious agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of application U.S. Ser.No. 08/286,430, filed Aug. 5, 1994, now U.S. Pat. No. 5,686,578.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel polyspecific immunoconjugatesthat are useful for diagnosis and therapy of diseases caused by cellsthat are multidrug resistant. In particular, this invention relates topolyspecific immunoconjugates that comprise at least one moiety thatbinds with a multidrug transporter protein, at least one moiety thatbinds with a tumor associated antigen or infectious agent antigen, and atherapeutic or diagnostic agent. This invention also relates to methodsof diagnosis and therapy using the polyspecific immunoconjugates. Thisinvention further relates to diagnostic and therapeutic uses of antibodycomposites comprising at least one moiety that binds with a multidrugtransporter protein, and at least one moiety that binds with a tumorassociated antigen or infectious agent antigen.

2. Background

One of the major limitations of cancer chemotherapy is the developmentof drug resistance by cancer cells. Despite initial sensitivity to aparticular chemotherapeutic agent, some tumors become progressivelyunresponsive to the particular agent, or to various chemotherapeuticagents. This phenomenon of acquired drug resistance is believed to bedue to the selection and growth of drug resistant mutant tumor cells.See, for example, Deuchars et al., Sem. Oncol. 16: 156 (1989).

Cultured cell lines and transplantable tumors have been used to studythe mechanism of acquired drug resistance in vitro. These studies haveshown that under certain selection conditions, cells may acquiresimultaneous resistance to a diverse group of drugs that are unrelatedto the selecting agent in structure, cellular target and mode of action.See, for example, Bradley et al., Biochim. Biophys. Acta 948: 87 (1988);Deuchars et al., supra. Many of the drugs affected by this“multidrug-resistance” (MDR) phenotype are important in currenttreatment protocols, such as vincristine, actinomycin D., andadriamycin. Id.

The MDR phenotype is consistently associated with over-expression of a170 kilodalton membrane glycoprotein, designated “gp170” or“P-glycoprotein.” Endicott et al., Ann. Rev. Biochem. 58: 137 (1989);Kane et al., J. Bioenerg. Biomembr. 22: 593 (1990); Efferth et al.,Urol. Res. 18: 309 (1990). Studies indicate that P-glycoprotein is atransmembrane protein responsible for an ATP-dependent efflux of a broadspectrum of structurally and functionally distinct drugs frommultidrug-resistant cells. Riordan et al., Pharmacol. Ther. 28: 51(1985). In fact, expression of P-glycoprotein has been shown to bepredictive of a poor response to chemotherapy in a number of neoplasms.See, for example, Pearson et al., J. Nat'l Cancer Inst. 83: 1386 (1991).

Recent observations indicate that infectious agents can induce the MDRphenotype in noncancerous cells. For example, prolonged treatment with3′-azido-3′-deoxythymidine (AZT) for human immunodeficiency virus (HIV)infection is associated with an acquired resistance to AZT. Gollapudi etal., Biochem. Biophys. Res. Commun. 171: 1002 (1990); Antonelli et al.,AIDS Research and Human Retroviruses 8: 1839 (1992). In vitro studiesdemonstrate that. HIV-infected human cells have an increased expressionof P-glycoprotein and accumulate less AZT, compared with non-infectedcontrol cells. Id.; Gupta et al., J. Clin. Immunol. 13: 289 (1993).Thus, overexpression of P-glycoprotein and the accompanying MDRphenotype can impair chemotherapy with anti-viral drugs.

Considerable effort has been employed to overcome themultidrug-resistant phenotype and thus, improve the efficacy ofchemotherapy. Most of these strategies have involved pharmacologicalagents that enhance the intracellular accumulation of the cancer drugsby biochemically inhibiting the multidrug transporter. See, for example,Ford et al., Pharmacol. Rev. 42: 155 (1990). Examples of agents thatmodulate P-glycoprotein activity include calcium channel blockers,calmodulin inhibitors, antiarrythmics, antimalarials, variouslysoosmotropic agents, steroids, antiestrogens, and cyclic peptideantibiotics. Rittmann-Grauer et al., Cancer Res. 52: 1810 (1992).

However, multidrug-resistant reversing drugs used in early clinicaltrials have shown major side effects unrelated to the inhibition ofP-glycoprotein, such as cardiac toxicity (verapamil) orimmunosuppression (cyclosporin A), which limit the dosage of drug thatcan be administered. See, for example, Ozols et al., J. Clin. Oncol. 5:641 (1987); Dalton et al., J. Clin. Oncol. 7: 415 (1989); Cano-Gauci etal., Biochem. Pharmacol. 36: 2115 (1987); Ford et al., supra. Thus,there has been limited success in reversing MDR in vivo due to thetoxicity of many of these small modulators. See, for example,Rittmann-Grauer et al., supra.

The use of antibody-drug conjugates provides an alternative approach toovercoming the MDR phenotype. For example, in vitro studies have shownthat MDR can be partially overcome by conjugating the resistant drug toan antitumor antibody to increase uptake and subsequent cell death.Durrant et al., Brit. J. Cancer 56: 722 (1987); Sheldon et al.,Anticancer Res. 9: 637 (1989). This approach, however, lacks specificityfor tumor cells that express the MDR phenotype.

A more targeted approach to overcoming the MDR phenotype is to useantibodies or antibody conjugates that bind with P-glycoprotein. Forexample, the administration of an anti-P-glycoprotein monoclonalantibody and a resistant drug can increase the survival time of nudemice that carry human tumor cells. Pearson et al., J. Nat'l Cancer Inst.83: 1386 (1991); Iwahashi et al., Cancer Res. 53: 5475 (1993). Also, seeGrauer et al., international publication No. WO 93/02105 (1993). Inaddition, an anti-P-glycoprotein monoclonal antibody-Pseudomonas toxinconjugate has been shown to kill multidrug-resistant human cells invitro. FitzGerald et al., Proc. Nat'l Acad. Sci. USA 84: 4288 (1987).Also, see Efferth et al., Med. Oncol. & Tumor Pharmacother. 9: 11(1992), and Mechetner et al., international publication No. WO 93/19094(1993).

Similarly, investigators have produced bispecific antibodies comprisinga P-glycoprotein binding moiety and a moiety that binds with a cytotoxiccell. van Dijk et al., Int. J. Cancer 44: 738 (1989); Ring et al.,international Publication No. WO 92/08802 (1992). The theory behind thisapproach is that the bispecific antibodies can be used to directcytotoxic cells to multidrug-resistant cells that expressP-glycoprotein.

However, studies have shown that P-glycoprotein is expressed in normalhuman tissues, such as liver, kidney, adrenal gland, pancreas, colon andjejunum. See, for example, Endicott et al., Ann. Rev. Biochem. 58: 137(1989). Consequently, investigators have warned that “blockingP-glycoprotein action in order to circumvent MDR will also affect thenormally expressed P-glycoprotein and this may cause unacceptable sidetoxic effects.” Childs et al., “The MDR Superfamily of Genes and ItsBiological Implications,” in IMPORTANT ADVANCES IN ONCOLOGY 1994, DeVitaet al., (eds.), pages 21-36 (J.B. Lippincott Co. 1994). This admonitionparticularly applies to therapeutic methods that use antibody conjugatesconsisting of a P-glycoprotein binding moiety and a cytotoxic agent.Therefore, the success of an antibody-directed treatment of MDR tumorswill mainly depend upon the ability to kill drug-resistant tumor cellswith tolerable side effects to normal tissues of the patient. Efferth etal., Med. Oncol. & Tumor Pharmacother. 9: 11 (1992).

Thus, an need exists for a method to overcome the MDR phenotype but thatalso minimizes toxicity to normal tissue.

The emergence of the MDR phenotype also is the major cause of failure inthe treatment of infectious diseases. Davies, Science 264: 375 (1994).In particular, pathogenic bacteria have active drug efflux systems ofvery broad substrate specificity. Nikaido, Science 264: 382 (1994),which is incorporated by reference. For example, studies indicate that adrug efflux system plays a major role in the intrinsic resistance ofPsuedomonas aeruginosa, a common opportunistic pathogen. Poole et al.,Mol. Microbiol. 10: 529 (1993); Poole et al., J. Bacteriol. 175: 7363(1993).

Recent studies indicate that bacterial drug efflux systems arefunctionally similar to the mammalian MDR efflux pump. As anillustration, both the Bacillus subtilis and the mammalian multidrugtransporters can be inhibited by reserpine and verapamil. Neyfakh etal., Proc. Nat'l Acad. Sci. 88: 4781 (1991). Moreover, investigatorshave recognized a superfamily of ATP-dependent membrane transportersthat includes prokaryotic permeases and mammalian P-glycoprotein. Doigeet al., Ann. Rev. Microbiol. 47: 291 (1993).

Active drug efflux as a mechanism for drug resistance is significant innonbacterial infectious agents. For instance, a Plasmodium falciparumprotein is involved in imparting resistance to quinoline-containingdrugs used for prophylaxis and treatment of malaria. Id.; Bray, FEMSMicrobiol. Lett. 113: 1 (1993). In addition, drug resistance has beenlinked to active efflux in the fungus, Aspergillus nidulans. de Waard etal., Pestic. Biochem. Physiol. 13: 255 (1980).

Historically, the pharmaceutical industry has concentrated on designingdrugs to overcome specific mechanisms of MDR in infectious agents, suchas increased degradation of particular drugs and inactivation of drugsby enzymatic modification of specific groups. Nikaido et al., supra.However, in the future, general mechanisms of MDR, such as active drugefflux, are likely to become more important in the clinical setting.

Thus, a need exists for methods that can be used to inhibit the functionof multidrug transporter proteins expressed by infectious agents.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide amethod for overcoming the multidrug-resistant phenotype that has atherapeutic index superior to conventional methods.

Another object of this invention is to provide methods for selectivelytargeting diagnostic and therapeutic agents to multidrug-resistantcells, while avoiding major toxic side effects to normal organs.

Another object of this invention is to provide antibody composites thatbind a multidrug transporter protein and an antigen associated with atumor or infectious agent.

A further object of this invention is to provide polyspecificimmunoconjugates which are conjugates of antibody composites anddiagnostic or therapeutic agents.

These and other objects are achieved, in accordance with one embodimentof the present invention by the provision of a polyspecificimmunoconjugate comprising:

(a) at least one antibody component that binds with a first epitope of amultidrug transporter protein;

(b) at least one antibody component that binds with a first epitope ofan antigen, wherein the antigen is associated with a tumor or aninfectious agent; and

(c) at least one diagnostic or therapeutic agent.

The antibody components of such a polyspecific immunoconjugate areselected from the group consisting of (a) a murine monoclonal antibody;(b) a humanized antibody derived from (a); (c) a human monoclonalantibody; (d) a subhuman primate antibody; and (e) an antibody fragmentderived from (a), (b), (c) or (d), wherein the antibody fragment isselected from the group consisting of F(ab′)₂, F(ab)₂, Fab′, Fab, Fv,sFv and minimal recognition unit. The multidrug transporter protein ofsuch a polyspecific immunoconjugate is selected from the groupconsisting of P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA,QacA, CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA,DrrA, DrrB, TlrC, Bmr, TetA and OprK.

As stated above, the polyspecific immunoconjugate comprises a diagnosticor therapeutic agent. A suitable diagnostic agent is selected from thegroup consisting of radioactive label, photoactive agent or dye,florescent label, enzyme label, bioluminescent label, chemiluminescentlabel, colloidal gold and paramagnetic ion. Moreover, a suitableradioactive label may be a γ-emitter or a positron-emitter. Preferably,γ-emitters have a gamma radiation emission peak in the range of 50-500Kev, such as a radioisotope selected from the group consisting of^(99m)Tc, ⁶⁷Ga, ¹²³I, ¹²⁵I and ¹³¹I.

A suitable therapeutic agent is selected from the group consisting ofradioisotope, boron addend, immunomodulator, toxin, photoactive agent ordye, cancer chemotherapeutic drug, antiviral drug, antifungal drug,antibacterial drug, antiprotozoal drug and chemosensitizing agent.Moreover a suitable therapeutic radioisotope is selected from the groupconsisting of α-emitters, β-emitters, γ-emitters, Auger electronemitters, neutron capturing agents that emit α-particles andradioisotopes that decay by electron capture. Preferably, theradioisotope is selected from the group consisting of ¹⁹⁸Au, ³²P, ¹²⁵I,¹³¹I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu and ²¹¹At.

The present invention also contemplates polyspecific immunoconjugateswhich further comprise an antibody component that binds with a secondepitope of the multidrug transporter protein. Moreover, polyspecificimmunoconjugates may additionally comprise an antibody component thatbinds with a second epitope of the tumor or infectious agent associatedantigen, or with an epitope of a second antigen associated with thetumor or the infectious agent.

The present invention also is directed to a method for treating a mammalhaving either a multidrug resistant tumor that expresses a tumorassociated antigen or a multidrug resistant disease caused by aninfectious agent, the method comprising the step of administering apolyspecific immunoconjugate to the mammal, wherein the polyspecificimmunoconjugate comprises:

(a) at least one antibody component that binds with a first epitope of amultidrug transporter protein,

(b) at least one antibody component that binds with a first epitope ofan antigen, wherein the antigen is associated with the tumor or theinfectious agent, and

(c) at least one therapeutic agent.

Moreover, the present invention contemplates methods further comprisingthe administration of a chemosensitizing agent or immunomodulator to themammal.

In addition, the present invention is directed to a method for detectingthe location of multidrug resistant (MDR) tumor cells, MDR HIV-infectedcells or MDR infectious agents in a mammal having a multidrug resistantdisease caused by a tumor or infectious agent, the method comprising thesteps of:

(a) parenterally injecting the mammal with an antibody compositecomprising (1) at least one antibody component that binds a firstepitope of a multidrug transporter protein, and (2) at least oneantibody component that binds a first epitope of an antigen that isassociated with the tumor or the infectious agent, wherein the antibodycomposite is conjugated with a biotin-binding molecule or with biotin;

(b) parenterally injecting a clearing composition comprised of:

(i) biotin, when the antibody composite is conjugated with abiotin-binding molecule, or

(ii) a biotin-binding molecule, when the antibody composite isconjugated with biotin,

and allowing the clearing composition to substantially clear theantibody composite from sites that do not contain MDR tumor cells, MDRHIV-infected cells or MDR infectious agents; and

(c) parenterally injecting a diagnostic composition comprised of:

(i) biotin, when the antibody composite is conjugated with abiotin-binding molecule, or

(ii) a biotin-binding molecule, when the antibody composite isconjugated with biotin,

and a diagnostic agent which is conjugated with the biotin or thebiotin-binding molecule.

In such a detection method, the diagnostic agent is selected from thegroup consisting of radioactive label, photoactive agent or dye,fluorescent label and paramagnetic ion. Moreover, the biotin-bindingmolecule is avidin or streptavidin.

The present invention also contemplates a method for treating a mammalhaving a multidrug resistant disease caused by a tumor or infectiousagent, the method comprising the steps of:

(a) parenterally injecting the mammal with an antibody compositecomprising (1) at least one antibody component that binds a firstepitope of a multidrug transporter protein, and (2) at least oneantibody component that binds a first epitope of an antigen that isassociated with the tumor or the infectious agent, wherein the antibodycomposite is conjugated with a biotin-binding molecule or with biotin;

(b) parenterally injecting a clearing composition comprised of:

(i) biotin, when the antibody composite is conjugated with abiotin-binding molecule, or

(ii) a biotin-binding molecule, when the antibody composite isconjugated with biotin,

and allowing the clearing composition to substantially clear theantibody composite from sites that do not contain multidrug resistant(MDR) cells or MDR infectious agents; and

(c) parenterally injecting a therapeutic composition comprised of:

(i) biotin, when the antibody composite is conjugated with abiotin-binding molecule, or

(ii) a biotin-binding molecule, when the antibody composite isconjugated with biotin,

and a therapeutic agent which is conjugated with the biotin or thebiotin-binding molecule.

A suitable therapeutic agent is selected from the group consisting ofradioisotope, boron addend, toxin, immunomodulator, photoactive agent ordye, cancer chemotherapeutic drug, antiviral drug, antifungal drug,antibacterial drug, antiprotozoal drug and a chemosensitizing agent.Again, the biotin-binding molecule is avidin or streptavidin.

The present invention also is directed to a method for detecting thepresence of multidrug resistant (MDR) tumor cells, MDR HIV-infectedcells or MDR infectious agents in a mammal, the method comprising:

(a) removing from the mammal a biological sample that is suspected ofcontaining MDR tumor cells, MDR HIV-infected cells or MDR infectiousagents;

(b) contacting the biological sample with an antibody composite whichcomprises (1) at least one antibody component that binds with a firstepitope of a multidrug transporter protein, and (2) at least oneantibody component that binds with a first epitope of an antigen that isassociated with the tumor or the infectious agent, wherein thecontacting is performed under conditions which allow the binding of theantibody composite to the biological sample; and

(c) detecting any of the bound antibody composite.

Here, a suitable diagnostic agent selected from the group consisting ofradioisotope, fluorescent label, chemiluminescent label, enzyme label,bioluminescent label and Colloidal gold. Moreover, the antibodycomposite can further comprise biotin or a biotin-binding molecule.

The present invention is further directed to a method for detecting thelocation of multidrug resistant (MDR) tumor cells, MDR HIV-infectedcells or MDR infectious agents in a mammal having a multidrug resistantdisease caused by a tumor or infectious agent, the method comprising thesteps of:

(a) parenterally injecting the mammal with a polyspecificimmunoconjugate that comprises (1) at least one antibody component thatbinds with a first epitope of a multidrug transporter protein, (2) atleast one antibody component that binds with a first epitope of anantigen that is associated with the tumor or infectious agent, and (3) adiagnostic agent;

(b) parenterally injecting the mammal with an antibody or antibodyfragment that binds with the polyspecific immunoconjugate in an amountthat is sufficient to decrease the level of circulating polyspecificimmunoconjugate by about 10-85% within 2 to 72 hours;

(c) scanning the mammal with a detector to locate the site or sites ofuptake of the polyspecific immunoconjugate.

A suitable diagnostic agent is selected from the group consisting ofradioactive label, photoactive agent or dye, fluorescent label andparamagnetic ion.

The present invention also contemplates a method for treating a mammalhaving a multidrug resistant disease caused by a tumor or infectiousagent, the method comprising the steps of:

(a) parenterally injecting the mammal with a polyspecificimmunoconjugate comprising (1) at least one antibody component thatbinds with a first epitope of a multidrug transporter protein, (2) atleast one antibody component that binds with a first epitope of anantigen that is associated with the tumor or infectious agent, and (3) atherapeutic agent; and

(b) parenterally injecting the mammal with an antibody or antibodyfragment that binds with the polyspecific immunoconjugate in an amountthat is sufficient to decrease the level of circulating polyspecificimmunoconjugate by about 10-85% within 2 to 72 hours.

In addition, the present invention is directed to a method for detectingthe location of multidrug resistant (MDR) tumor cells, MDR HIV-infectedcells or MDR infectious agents in a subject having a multidrug resistantdisease caused by a tumor or infectious agent, the method comprising thesteps of:

(a) parenterally injecting the subject with a polyspecificimmunoconjugate comprising (1) at least one antibody component thatbinds with a first epitope of a multidrug transporter protein, (2) atleast one antibody component that binds with a first epitope of anantigen that is associated with a tumor or infectious agent, and (3) adiagnostic agent;

(b) surgically exposing or endoscopically accessing the interior of thebody cavity of the subject; and

(c) scanning , the interior body cavity with a detection probe to detectthe sites of accretion of the polyspecific immunoconjugate.

Suitable diagnostic agents include radioisotopes, such as a γ-emitter ora positron-emitter, and a photoactive agent or dye that is detected bylaser-induced fluorescence.

The present invention also contemplates a method for treating a subjecthaving a multidrug resistant disease caused by a tumor or infectiousagent, the method comprising the steps of:

(a) parenterally injecting the subject with a polyspecificimmunoconjugate comprising (1) at least one antibody component thatbinds with a first epitope of a multidrug transporter protein, (2) atleast one antibody component that binds with a first epitope of anantigen that is associated with a tumor or infectious agent, and (3) aphotoactive agent or dye;

(b) surgically exposing or endoscopically accessing the interior of thebody cavity of the subject; and

(c) treating sites of accretion of the polyspecific immunoconjugate tolight, wherein the treatment activates the photoactive agent or dye.

In addition, the present invention is directed to an antibody compositecomprising:

(a) at least one antibody component that binds with a first epitope of amultidrug transporter protein; and

(b) at least one antibody component that binds with a first epitope ofan antigen, wherein the antigen is associated with a tumor or aninfectious agent.

Suitable antibody components of antibody composites are selected fromthe group consisting of (a) a murine monoclonal antibody; (b) ahumanized antibody derived from (a); (c) a human monoclonal antibody;(d) a subhuman primate antibody; and (e) an antibody fragment derivedfrom (a), (b), (c) or (d), where an antibody fragment is selected fromthe group consisting of F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv and minimalrecognition unit. Moreover, a suitable multidrug transporter protein isselected from the group consisting of P-glycoprotein, OtrB, Tel(L), Mmr,ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr,QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr, TetA and OprK.

The present invention also contemplates an antibody composite furthercomprising an antibody component that binds with a second epitope of themultidrug transporter protein. An antibody composite can additionallyinclude an antibody component that binds with a second epitope of thetumor or infectious agent associated antigen, or with an epitope of asecond antigen associated with the tumor or the infectious agent.

The present invention is further directed to a method for treating amammal having either a multidrug resistant tumor that expresses a tumorassociated antigen or a multidrug resistant disease caused by aninfectious agent, the method comprising the step of administering anantibody composite to the mammal, wherein the antibody compositecomprises:

(a) at least one antibody component that binds with a first epitope of amultidrug transporter protein, and

(b) at least one antibody component that binds with a first epitope ofan antigen, wherein the antigen is associated with the tumor or theinfectious agent.

Moreover, the present invention contemplates a method further comprisingthe step of administering a therapeutic agent to the mammal, wherein thetherapeutic agent is selected from the group consisting of cancerchemotherapeutic drug, antiviral drug, antifungal drug, antibacterialdrug and antiprotozoal drug. Finally, the present invention also isdirected to a method which further comprises the step of administeringan immunomodulator, wherein the immunomodulator is selected from thegroup consisting of cytokine, stem cell growth factor and hematopoieticfactor.

DETAILED DESCRIPTION

1. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

A structural gene is a DNA sequence that is transcribed into messengerRNA (MRNA) which is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

A promoter is a DNA sequence that directs the transcription of astructural gene. Typically, a promoter is located in the 5′ region of agene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. In contrast, the rate oftranscription is not regulated by an inducing agent if the promoter is aconstitutive promoter.

An isolated DNA molecule is a fragment of DNA that is not integrated inthe genomic DNA of an organism. For example, a cloned T cell receptorgene is a DNA fragment that has been separated from the genomic DNA of amammalian cell. Another example of an isolated DNA molecule is achemically-synthesized DNA molecule that is not integrated in thegenomic DNA of an organism.

An enhancer is a DNA regulatory element that can increase the efficiencyof transcription, regardless of the distance or orientation of theenhancer relative to the start site of transcription.

Complementary DNA (cDNA) is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of MRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand.

The term expression refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage, that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance.

An expression vector is a DNA molecule comprising a gene that isexpressed in ma host cell. Typically, gene expression is placed underthe control of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

A tumor associated antigen is a protein normally not expressed, orexpressed at very low levels, by a normal counterpart. Examples of tumorassociated antigens include α-fetoprotein and carcinoembryonic antigen(CEA). Many other illustrations of tumor associated antigens are knownto those of skill in the art. See, for example, Urban et al., Ann. Rev.Immunol. 10: 617 (1992).

As used herein, an infectious agent denotes both microbes and parasites.A “microbe” includes viruses, bacteria, rickettsia, mycoplasma,protozoa, fungi and like microorganisms. A “parasite” denotesinfectious, generally microscopic or very small multicellularinvertebrates, or ova or juvenile forms thereof, which are susceptibleto antibody-induced clearance or lytic or phagocytic destruction, suchas malarial parasites, spirochetes, and the like.

A multidrug transporter protein is a membrane-associated protein whichtransports diverse cytotoxic compounds out of a cell in anenergy-dependent manner. Examples of multidrug transporter proteinsinclude P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA,CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA,DrrB, TlrC, Bmr, TetA, OprK, and the like.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody.

The term “antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“sFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

Humanized antibodies are recombinant proteins in which murinecomplementary determining regions of monoclonal antibodies have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

As used herein, the term antibody component includes both an entireantibody and an antibody fragment.

As used herein, a diagnostic or therapeutic agent is a molecule or atomwhich is conjugated to an antibody moiety to produce a conjugate whichis useful for diagnosis or for therapy. Examples of diagnostic ortherapeutic agents include drugs, toxins, immunomodulators, chelators,boron compounds, photoactive agents or dyes, radioisotopes, fluorescentagents, paramagnetic ions or molecules and marker moieties.

An antibody composite is a polyspecific antibody composition comprisingat least two substantially monospecific antibody components, wherein atleast one antibody component binds with an epitope of a multidrugtransporter protein, and wherein at least one antibody component bindswith an antigen that is associated with either a tumor or an infectiousagent.

A volyspecific immunoconiugate is a conjugate of an antibody compositewith a diagnostic or therapeutic agent.

2. Production of Rodent Monoclonal Antibodies, Humanized Antibodies,Primate Antibodies and Human Antibodies

An antibody composite of the present invention may be derived from arodent monoclonal antibody (MAb) Rodent monoclonal antibodies tospecific antigens may be obtained by methods known to those skilled inthe art. See, for example, Kohler and Milstein, Nature 256: 495 (1975),and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1,pages 2.5.1-2.6.7 (John Wiley & Sons 1991) [hereinafter “Coligan”].Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising an antigen, verifying the presence of antibodyproduction by removing a serum sample, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

A wide variety of monoclonal antibodies against tumor associatedantigens or infectious agents have been developed. See, for example,Goldenberg et al., international application publication No. WO91/11465(1991), Hansen et al., international application publication No.WO 93/23062, and Goldenberg, international application publication No.WO 94/04702 (1994), each of which is incorporated by reference in itsentirety.

Furthermore, such antibodies are readily available from commercialsources. For example, rodent monoclonal antibodies that bind withadenocarcinoma-associated antigen (Cat. No. 121730), human chorionicgonadotropin (Cat. No. 230740), carcinoembryonic antigen (Cat. Nos.215920 and 215922), human alpha-fetoprotein (Cat. No. 341646), and thelike can be obtained from CalbiochemNovabiochem Corp. (San Diego,Calif.). Moreover, rodent monoclonal antibodies that bind with antigenicdeterminants of infectious agents such as Escherichia coli (HB 8178),Legionella pneumophila (CRL 1770), Schistosoma mansoni (HB 8088),Streptococcus, Group A (HB 9696), Treponema pallidum (HB 8134),hepatitis B (CRL 8017), herpes simplex (HB 8181), humanimmunodeficiency. virus (HB 9101), among others, can be obtained fromAmerican Type Culture Collection (Rockville, Md.). Furthermore, murinemonoclonal antibodies against merozoites and sporozoites of Plasmodiumfalciparum can be prepared as described by Goldenberg, U.S. Pat. No.5,332,567 (1994), which is incorporated by reference.

Methods for producing P-glycoprotein antibodies are well-known to thoseof skill in the art. See, for example, Lathan et al., Cancer Res. 45:5064 (1985); Kartner et al., Nature 316: 820 (1985); Hamada et al.,Proc. Nat'l Acad. Sci 83: 7785 (1986); Scheper et al., Int. J. Cancer42: 389 (1988); Rittmann-Grauer et al., Cancer Res. 52: 1810 (1992);Ling et al., U.S. Pat. No. 4,837,306 (1989); Ring et al., internationalpublication No. WO 92/08802; Grauer et al., international publicationNo. WO 93/02105; and Mechetner et al., international publication No. WO93/19094, which are incorporated by reference. Since P-glycoproteinretains its structural identity across different mammalian species(Rubin, U.S. Pat. No. 5,005,588; Kane et al., J. Bioenergetics andBiomembranes 22: 593 (1990)), antibodies raised against P-glycoproteinfrom non-human cells can be used for diagnosis and therapy in humans.Conversely, antibodies raised against human P-glycoprotein should besuitable for veterinary uses.

Preferred P-glycoprotein antibodies bind with the extracellular domainof P-glycoprotein, and can be produced against cells that express theMDR phenotype as described, for example, by Mechetner et al., supra, andRittmann-Grauer et al., supra. Alternatively, such antibodies can beobtained using peptides that contain an extracellular epitopeP-glycoprotein. See, for example, Cianfriglia et al., internationalpublication No. WO 93/25700, which is incorporated by reference.

Those of skill in the art can readily apply standard techniques toproduce antibodies against multidrug transporter proteins of infectiousagents. Suitable antigens include multidrug transporter proteins such asBmr, TetA, EmrB, OprK, Smr, and the like. See, for example, Nikaido etal., supra; Poole et al., J. Bacteriol. 175: 7363 (1993); and Childs etal., “The MDR Superfamily of Genes and Its Biological Implications,” inIMPORTANT ADVANCES IN ONCOLOGY 1994, DeVita et al., (eds.), pages 21-36(J.B. Lippincott Co. 1994), which are incorporated by reference. Oneapproach for preparing antibodies against infectious agent multidrugtransporter proteins is illustrated in Example 6.

An antibody composite of the present invention may also be derived froma subhuman primate antibody. General techniques for raisingtherapeutically useful antibodies in baboons may be found, for example,in Goldenberg et al., international patent publication No. WO 91/11465(1991), and in Losman et al., Int. J. Cancer 46: 310 (1990), which isincorporated by reference.

Alternatively, an antibody composite may be derived from a “humanized”monoclonal antibody. Humanized monoclonal antibodies are produced bytransferring mouse complementary determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain, and then, substituting human residues in the framework regionsof the murine counterparts. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith the immunogenicity of murine constant regions. General techniquesfor cloning murine immunoglobulin variable domains are described, forexample, by the publication of Orlandi et al., Proc. Nat'l Acad. Sci.USA 86: 3833 (1989), which is incorporated by reference in its entirety.Techniques for producing humanized MAbs are described, for example, byJones et al., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323(1988), Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc.Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), and Singer et al., J. Immun. 150: 2844 (1993), each of whichis hereby incorporated by reference.

As an alternative, an antibody composite of the present invention may bederived from human antibody fragments isolated from a combinatorialimmunoglobulin library. See, for example, Barbas et al., METHODS: Acompanion to Methods in Enzymology 2: 119 (1991), and Winter et al.,Ann. Rev. Immunol. 12: 433 (1994), which are incorporated by reference.Cloning and expression vectors that are useful for producing a humanimmunoglobulin phage library can be obtained, for example, fromSTRATAGENE Cloning Systems (La Jolla, Calif.).

In addition, an antibody composite of the present invention may bederived from a human monoclonal antibody. Such antibodies are obtainedfrom transgenic mice that have been “engineered” to produce specifichuman antibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain locus are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci.The transgenic mice can synthesize human antibodies specific for humanantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7: 13 (1994), Lonberg etal., Nature 368: 856 (1994), and Taylor et al., Int. Immun. 6: 579(1994), which are incorporated by reference.

3. Production of Antibody Fragments

The present invention contemplates the use of antibody fragments toproduce antibody composites. Antibody fragments can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofthe DNA coding for the fragment. Antibody fragments can be obtained bypepsin or papain digestion of whole antibodies by conventional methods.For example, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein, which patents areincorporated herein in their entireties by reference. Also, see Nisonoffet al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73:119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described in Inbar etal., Proc. Nat'l Acad. Sci. USA 69: 2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcrosslinked by chemicals such as glutaraldehyde. See, for example,Sandhu, supra.

Preferably, the Fv fragments comprise VH and VL chains which areconnected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingDNA sequences encoding the V_(H) and V_(L) domains which are connectedby an oligonucleotide. The structural gene is inserted into anexpression vector which is subsequently introduced into a host cell,such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow etal., Methods: A Companion to Methods in Enzymology 2: 97 (1991). Alsosee Bird et al., Science 242:423-426 (1988), Ladner et al., U.S. Pat.No. 4,946,778, Pack et al., Bio/Technology 11:1271-1277 (1993), andSandhu, supra.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2: 106 (1991).

4. Production of Antibody composites

Antibody composites can be prepared by a variety of conventionalprocedures, ranging from glutaraldehyde linkage to more specificlinkages between functional groups. The antibodies and/or antibodyfragments are preferably covalently bound to one another, directly orthrough a linker moiety, through one or more functional groups on theantibody or fragment, e.g., amine, carboxyl, phenyl, thiol, or hydroxylgroups. Various conventional linkers in addition to glutaraldehyde canbe used, e.g., disiocyanates, diiosothiocyanates,bis(hydroxysuccinimide) esters, carbodiimides,maleimidehydroxysuccinimde esters, and the like. The optimal length ofthe linker may vary according to the type of target cell. The mostefficacious linker size can be determined by using antibody compositeswith various linker lengths for the immunochemical staining of a patienttissue sample that contains cells expressing a multidrug transporterprotein and the target antigen. Immunochemical techniques are describedbelow.

A simple method to produce antibody composites is to mix the antibodiesor fragments in the presence of glutaraldehyde to form an antibodycomposite. The initial Schiff base linkages can be stabilized, e.g., byborohydride reduction to secondary amines. A diiosothiocyanate orcarbodiimide can be used in place of glutaraldehyde as anon-site-specific linker.

The simplest form of an antibody composite is a bispecific antibodycomprising binding moieties for a multidrug transporter protein and anantigen that is associated with a tumor cell or infectious agent.Bispecific antibodies can be made by a variety of conventional methods,e.g., disulfide cleavage and reformation of mixtures of whole IgG or,preferably F(ab′)₂ fragments, fusions of more than one hybridoma to formpolyomas that produce antibodies having more than one specificity, andby genetic engineering. Bispecific antibody composites have beenprepared by oxidative cleavage of Fab′ fragments resulting fromreductive cleavage of different antibodies. This is advantageouslycarried out by mixing two different F(ab′)₂ fragments produced by pepsindigestion of two different antibodies, reductive cleavage to form amixture of Fab′ fragments, followed by oxidative reformation of thedisulfide linkages to produce a mixture of F(ab′)₂ fragments includingbispecific antibody composites containing a Fab′ potion specific to eachof the original epitopes. General techniques for the preparation ofantibody composites may be found, for example, in Nisonhoff et al., ArchBiochem. Biophys. 93: 470 (1961), Hammerling et al., J. Exp. Med. 128:1461 (1968), and U.S. Pat. No. 4,331,647.

More selective linkage can be achieved by using a heterobifunctionallinker such as maleimidehydroxysuccinimide ester. Reaction of the esterwith an antibody or fragment will derivatize amine groups on theantibody or fragment, and the derivative can then be reacted with, e.g.,an antibody Fab fragment having free sulfhydryl groups (or, a largerfragment or intact antibody with sulfhydryl groups appended thereto by,e.g., Traut's Reagent). Such a linker is less likely to crosslink groupsin the same antibody and improves the selectivity of the linkage.

It is advantageous to link the antibodies or fragments at sites remotefrom the antigen binding sites. This can be accomplished by, e.g.,linkage to cleaved interchain sulfydryl groups, as noted above. Anothermethod involves reacting an antibody having an oxidized carbohydrateportion with another antibody which has at lease one free aminefunction. This results in an initial Schiff base (imine) linkage, whichis preferably stabilized by reduction to a secondary amine, e.g., byborohydride reduction, to form the final composite. Such site-specificlinkages are disclosed, for small molecules, in U.S. Pat. No. 4,671,958,and for larger addends in U.S. Pat. No. 4,699,784.

In the present context, a bispecific antibody comprises binding moietiesfor a multidrug transporter protein and an antigen that is associatedwith a tumor cell or infectious agent. For example, the multidrugtransporter protein-binding moiety can be derived from anti-multidrugtransporter protein Mab, while a carcinoembryonic antigen (CEA) bindingmoiety can be derived from a Class III Mab. Methods for preparingmultidrug transporter protein Mab are described above, while methods forpreparing Class III anti-CEA Mab are described by Primus et al., CancerResearch 43: 686 (1983), and by Primus et al., U.S. Pat. No. 4,818,709,which are incorporated by reference.

For example, a bispecific antibody can be prepared by obtaining anF(ab′)₂ fragment from an anti-CEA Class III Mab, using the techniquesdescribed above. The interchain disulfide bridges of the anti-CEA ClassIII F(ab′)₂ fragment are gently reduced with cysteine, taking care toavoid light-heavy chain linkage, to form Fab′-SH fragments. The SHgroup(s) is(are) activated with an excess of bis-maleimide linker(1,1′-(methylenedi-4,1-phenylene)bis-malemide). The multidrugtransporter protein Mab is converted to Fab′-SH and then reacted withthe activated anti-CEA Class III Fab′-SH fragment to obtain a bispecificantibody.

Alternatively, such bispecific antibodies can be produced by fusing twohybridoma cell lines that produce anti-multidrug transporter protein Maband anti-CEA Class III Mab. Techniques for producing tetradomas aredescribed, for example, by Milstein et al., Nature 305: 537 (1983) andPohl et al., Int. J. Cancer 54: 418 (1993).

Finally, such bispecific antibodies can be produced by geneticengineering. For example, plasmids containing DNA coding for variabledomains of an anti-CEA Class III Mab can be introduced into hybridomasthat secrete anti-multidrug transporter protein antibodies. Theresulting “transfectomas”produce bispecific antibodies that bind CEA andthe multidrug transporter protein. Alternatively, chimeric genes can bedesigned that encode both anti-multidrug transporter protein andanti-CEA binding domains. General techniques for producing bispecificantibodies by genetic engineering are described, for example, bySongsivilai et al., Biochem. Biophys. Res. Commun. 164: 271 (1989);Traunecker et al., EMBO J. 10: 3655 (1991); and Weiner et al., J.Immunol. 147: 4035 (1991).

A polyspecific antibody composite can be obtained by adding variousantibody components to a bispecific antibody composite. For example, abispecific antibody can be reacted with 2-iminothiolane to introduce oneor more sulfhydryl groups for use in coupling the bispecific antibody toan antibody component that binds an epitope of a multidrug transporterprotein that is distinct from the epitope bound by the bispecificantibody, using the bis-maleimide activation procedure described above.These techniques for producing antibody composites are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,925,648,and Goldenberg, international publication No. WO 92/19273, which areincorporated by reference.

5. Preparation of Polyspecific Immunoconjugates

Polyspecific immunoconjugates can be prepared by indirectly conjugatinga diagnostic or therapeutic agent to an antibody composite. Generaltechniques are described in Shih et al., Int. J. Cancer 41:832-839(1988); Shih et al., Int. J. Cancer 46:1101-1106 (1990); and Shih etal., U.S. Pat. No. 5,057,313. The general method involves reacting anantibody component having an oxidized carbohydrate portion with acarrier polymer that has at least one free amine function and that isloaded with a plurality of drug, toxin, chelator, boron addends, orother diagnostic or therapeutic agent. This reaction results in aninitial Schiff base (imine) linkage, which can be stabilized byreduction to a secondary amine to form the final conjugate.

The carrier polymer -is preferably an aminodextran or polypeptide of atleast 50 amino acid residues, although other substantially equivalentpolymer carriers can also be used. Preferably, the final polyspecificimmunoconjugate is soluble in an aqueous solution, such as mammalianserum, for ease of administration and effective targeting for use indiagnosis or therapy. Thus, solubilizing functions on the carrierpolymer will enhance the serum solubility of the final polyspecificimmunoconjugate. Solubilizing functions also are important for use ofpolyspecific immunoconjugates for immunochemical detection, as describedbelow. In particular, an aminodextran will be preferred.

The process for preparing a polyspecific immunoconjugate with anaminodextran carrier typically begins with a dextran polymer,advantageously a dextran of average molecular weight of about10,000-100,000. The dextran is reacted with an oxidizing agent to effecta controlled oxidation of a portion of its carbohydrate rings togenerate aldehyde groups. The oxidation is conveniently effected withglycolytic chemical reagents such as NaIO₄, according to conventionalprocedures.

The oxidized dextran is then reacted with a polyamine, preferably adiamine, and more preferably, a mono- or polyhydroxy diamine. Suitableamines include ethylene diamine, propylene diamine, or other likepolymethylene diamines, diethylene triamine or like polyamines,1,3-diamino-2-hydroxypropane, or other like hydroxylated diamines orpolyamines, and the like. An excess of the amine relative to thealdehyde groups of the dextran is used to insure substantially completeconversion of the aldehyde functions to Schiff base groups.

A reducing agent, such as NaBH₄, NaBH₃CN or the like, is used to effectreductive stabilization of the resultant Schiff base intermediate. Theresultant adduct can be purified by passage through a conventionalsizing column to remove cross-linked dextrans.

Other conventional methods of derivatizing a dextran to introduce aminefunctions can also be used, e.g., reaction with cyanogen bromide,followed by reaction with a diamine.

The aminodextran is then reacted with a derivative of the particulardrug, toxin, chelator, paramagnetic ion, boron addend, or otherdiagnostic or therapeutic agent to be loaded, in an activated form,preferably, a carboxyl-activated derivative, prepared by conventionalmeans, e.g., using dicyclohexylcarbodiimide (DCC) or a water solublevariant thereof, to form an intermediate adduct.

Alternatively, polypeptide toxins such as pokeweed antiviral protein orricin A-chain, and the like, can be coupled to aminodextran byglutaraldehyde condensation or by reaction of activated carboxyl groupson the protein with amines on the aminodextran.

Chelators for radiometals or magnetic resonance enhancers are well-knownin the art. Typical are derivatives of ethylenediaminetetraacetic acid(EDTA) and diethylenetriaminepentaacetic acid (DTPA). These chelatorstypically have groups on the side chain by which the chelator can beattached to a carrier. Such groups include, e.g., benzylisothiocyanate,by which the DTPA or EDTA can be coupled to the amine group of acarrier. Alternatively, carboxyl groups or amine groups on a chelatorcan be coupled to a carrier by activation or prior derivatization andthen coupling, all by wellknown means.

Labels such as enzymes, fluorescent compounds, electron transfer agents,and the like can be linked to a carrier by conventional methods wellknown to the art. These labeled carriers and the polyspecificimmunoconjugates prepared from them can be used for immunochemicaldetection, as described below.

Boron addends, such as carboranes, can be attached to antibodycomponents by conventional methods. For example, carboranes can beprepared with carboxyl functions on pendant side chains, as is wellknown in the art. Attachment of such carboranes to a carrier, e.g.,aminodextran, can be achieved by activation of the carboxyl groups ofthe carboranes and condensation with amines on the carrier to produce anintermediate conjugate. Such intermediate conjugates are then attachedto antibody components to produce therapeutically useful polyspecificimmunoconjugates, as described below.

A polypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier must have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,chelator, boron addend or other diagnostic or therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, copolymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andpolyspecific immunoconjugate.

Conjugation of the intermediate conjugate with the antibody component iseffected by oxidizing the carbohydrate portion of the antibody componentand reacting the resulting aldehyde (and ketone) carbonyls with aminegroups remaining on the carrier after loading with a drug, toxin,chelator, boron addend, or other diagnostic or therapeutic agent.Alternatively, an intermediate conjugate can be attached to an oxidizedantibody component via amine groups that have been introduced in theintermediate conjugate after loading with the diagnostic or therapeuticagent. oxidation is conveniently effected either chemically, e.g., withNaIO₄ or other glycolytic reagent, or enzymatically, e.g., withneuraminidase and galactose oxidase. In the case of an aminodextrancarrier, not all of the amines of the aminodextran are typically usedfor loading a diagnostic or therapeutic agent. The remaining amines ofaminodextran condense with the oxidized antibody component to formSchiff base adducts, which are then reductively stabilized, normallywith a borohydride reducing agent.

Analogous procedures are used to produce other polyspecificimmunoconjugates according to the invention. Loaded polypeptide carrierspreferably have free lysine residues remaining for condensation with theoxidized carbohydrate portion of an antibody component. Carboxyls on thepolypeptide carrier can, if necessary, be converted to amines by, e.g.,activation with DCC and reaction with an excess of a diamine.

The final polyspecific immunoconjugate is purified using conventionaltechniques, such as sizing chromatography on Sephacryl S-300.

Alternatively, polyspecific immunoconjugates can be prepared by directlyconjugating an antibody component with a diagnostic or therapeuticagent. The general procedure is analogous to the indirect method ofconjugation except that a diagnostic or therapeutic agent is directlyattached to an oxidized antibody component.

It will be appreciated that other diagnostic or therapeutic agents canbe substituted for the chelators described herein. Those of skill in theart will be able to devise conjugation schemes without undueexperimentation.

In addition, those of skill in the art will recognize numerous possiblevariations of the conjugation methods. For example, the carbohydratemoiety can be used to attach polyethyleneglycol in order to extend thehalflife of an intact antibody, or antigen-binding fragment thereof, inblood, lymph, or other extracellular fluids. Moreover, it is possible toconstruct a “divalent immunoconjugate” by attaching a diagnostic ortherapeutic agent to a carbohydrate moiety and to a free sulfhydrylgroup. Such a free sulfhydryl group may be located in the hinge regionof the antibody component.

6. Use of Polyspecific Immunoconjugates and Antibody Composites forDiagnosis

A. In Vitro Diagnosis

The present invention contemplates the use of polyspecificimmunoconjugates and antibody composites to screen biological samples invitro for the expression of P-glycoprotein by tumor cells. For example,the polyspecific immunoconjugates and antibody composites of the presentinvention can be used to detect the presence of P-glycoprotein and tumorassociated antigen in tissue sections prepared from a biopsy specimen.Such immunochemical detection can be used to determine the abundance ofP-glycoprotein and to determine the distribution of P-glycoprotein inthe examined tissue. General immunochemistry techniques are well-knownto those of ordinary skill. See, for example, Ponder, “Cell MarkingTechniques and Their Application,” in MAMMALIAN DEVELOPMENT: A PRACTICALAPPROACH, Monk (ed.), pages 115-38 (IRL Press 1987), Volm et al., Eur.J. Cancer Clin. Oncol. 25: 743 (1989), Coligan at pages 5.8.1-5.8.8, andAusubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages14.6.1 to 14.6.13 (Wiley Interscience 1990). Also, see generally, Manson(ed.), METHODS IN MOLECULAR BIOLOGY, VOL.10: IMMUNOCHEMICAL PROTOCOLS(The Humana Press, Inc. 1992). Moreover, methods for the immunochemicaldetection of P-glycoprotein are described, for example, by Dalton etal., Blood 73: 747 (1989), and Volm et al., Eur. J. Cancer Clin. Oncol.25: 743 (1989).

In addition, the present invention contemplates the use of polyspecificimmunoconjugates and antibody composites to screen biological samples invitro for the expression of a multidrug transporter protein by aninfectious agent. For example, the polyspecific immunoconjugates andantibody composites of the present invention can be used to detect thepresence of OprK protein in clinical isolates. The presence of thisparticular multidrug transporter protein would indicate that the tissuewas infected with multidrug resistant Psuedomonas aeruginosa.

Moreover, immunochemical detection techniques can be used to optimizeantibody composites for subsequent in vivo diagnosis and therapy in theform of antibody composites per se or as polyspecific immunoconjugates.Accordingly, immunochemical detection can be performed with a battery ofantibody composites to identify the most appropriate combination ofantibody components for subsequent in vivo diagnosis and therapy. Forexample, an antibody moiety that binds the c-erb B2 proto-oncogeneproduct may be more suitable for a particular breast cancer than anantibody moiety that binds carcinoembryonic antigen. After a suitablecombination of antibody components have been identified, further invitro testing can be used to delineate the most efficacious linker sizein the antibody composite, as discussed above.

Immunochemical detection can be performed by contacting a biologicalsample with an antibody composite and then contacting the biologicalsample with a detectably labeled molecule which binds to the antibodycomposite. For example, the detectably labeled molecule can comprise anantibody moiety that binds the antibody composite. Alternatively, theantibody composite can be conjugated with avidin/streptavidin (orbiotin) and the detectably labeled molecule can comprise biotin (oravidin/streptavidin). Numerous variations of this basic technique arewell-known to those of skill in the art.

Alternatively, an antibody composite can be conjugated with a diagnosticagent to form a polyspecific immunoconjugate. Antibody composites can bedetectably labeled with any appropriate marker moiety, for example, aradioisotope, a fluorescent label, a chemiluminescent label, an enzymelabel, a bioluminescent label or colloidal gold. Methods of making anddetecting such detectably-labeled polyspecific immunoconjugates arewell-known to those of ordinary skill in the art, and are described inmore detail below.

The marker moiety can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Polyspecific immunoconjugates also can be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody component isdetermined by exposing the polyspecific immunoconjugate to light of theproper wavelength and detecting the resultant fluorescence. Fluorescentlabeling compounds include fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

Alternatively, polyspecific immunoconjugates can be detectably labeledby coupling an antibody component to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged polyspecific immunoconjugate isdetermined by detecting the presence of luminescence that arises duringthe. course of a chemical reaction. Examples of chemiluminescentlabeling compounds include luminol, isoluminol, an aromatic acridiniumester, an imidazole, an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label polyspecificimmunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, polyspecific immunoconjugates can be detectably labeledby linking an antibody component to an enzyme. When the polyspecificimmunoconjugates-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels which canbe employed in accordance with the present invention. The binding ofmarker moieties to antibody components can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al., Clin. Chim. Acta 70: 1 (1976), Schurs etal., Clin. Chim. Acta 81: 1 (1977), Shih et al., Int'l J. Cancer 46:1101 (1990), Stein et al., Cancer Res. 50: 1330 (1990), supra, and Steinet al., Int. J. Cancer 55: 938 (1993). Also, see generally, Coligan.

In addition, the convenience and versatility of immunochemical detectioncan be enhanced by using antibody components that have been conjugatedwith avidin, streptavidin, and biotin. See, for example, Wilchek et al.(eds.), Avidin-Biotin Technology, METHODS IN ENZYMOLOGY, VOL. 184(Academic Press 1990), and Bayer et al., “Immunochemical Applications ofAvidin-Biotin Technology,” in METHODS IN MOLECULAR BIOLOGY, VOL. 10,Manson (ed.), pages 149-162 (The Human Press, Inc. 1992).

Thus, the above-described immunochemical detection methods can be usedto assist in the diagnosis or staging of a pathological condition. Thesetechniques also can be used to identify the most suitable composition ofantibody composite or polyspecific immunoconjugate for subsequent invivo diagnosis and therapy.

B. In Vivo Diagnosis

The present invention also contemplates the use of antibody compositesand polyspecific immunoconjugates for in vivo diagnosis. The method ofdiagnostic imaging with radiolabeled MAbs is well-known. In thetechnique of immunoscintigraphy, for example, antibodies are labeledwith a gamma-emitting radioisotope and introduced into a patient. Agamma camera is used to detect the location and distribution ofgamma-emitting radioisotopes. See, for example, Srivastava (ed.),RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY (Plenum Press1988), Chase, “Medical Applications of Radioisotopes,” in REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al. (eds.), pp.624-652 (Mack Publishing Co., 1990), Brown, “Clinical Use of MonoclonalAntibodies,” in BIOTECHNOLOGY AND PHARMACY 227-49, Pezzuto et al. (eds.)(Chapman & Hall 1993), and Goldenberg, CA—A Cancer Journal forClinicians 44: 43 (1994).

For diagnostic imaging, radioisotopes may be bound to an antibodycomposite either directly, or indirectly by using an intermediaryfunctional group. Useful intermediary functional groups includechelators such as ethylenediaminetetraacetic acid anddiethylenetriaminepentaacetic acid. For example, see Shih et al., supra,and U.S. Pat. No. 5,057,313. Also, see Griffiths, U.S. Pat. No.5,128,119 (1992).

The radiation dose delivered to the patient is maintained at as low alevel as possible through the choice of isotope for the best combinationof minimum half-life, minimum retention in the body, and minimumquantity of isotope which will permit detection and accuratemeasurement. Examples of radioisotopes that can be bound to antibodycomposites and are appropriate for diagnostic imaging include γ-emittersand positron-emitters such as ^(99m)Tc, ⁶⁷Ga, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ⁵¹Co, ⁸⁹Zr, ¹⁸F and ⁶⁸Ga. Other suitable radioisotopes are knownto those of skill in the art.

Preferred γ-emitters have a gamma radiation emission peak in the rangeof 50-500 Kev, primarily because the state of the art for radiationdetectors currently favors such labels. Examples of such γ-emittersinclude ^(99m)Tc, ⁶⁷Ga, ¹²³I, ¹²⁵I and ¹³¹I.

Antibody composites also can be labeled with paramagnetic ions forpurposes of in vivo diagnosis. Elements that are particularly useful formagnetic resonance imaging include Gd, Mn, Dy and Fe ions.

A high background level of non-targeted antibody provides a majorimpediment to in vivo diagnosis methodology. However, the ratio oftarget to nontarget radiolabeled antibody can be enhanced through theuse of a nonlabeled second antibody which scavenges and promotes theclearance of the nontargeted circulating radiolabeled antibody. Thesecond antibody may be whole IgG or IgM, or a fragment of IgG or IgM, solong as it is capable of binding the radiolabeled antibody to form acomplex which is cleared from the circulation and nontarget spaces morerapidly than the radiolabeled antibody alone. In the present context,suitable second antibodies may bind with either the Fc portion orvariable region of a radiolabeled polyspecific immunoconjugate. See, forexample, Goldenberg, U.S. Pat. No. 4,624,846, Goldenberg, internationalpublication No. WO 92/19273, and Sharkey et al., Int. J. Cancer 51: 266(1992), which are incorporated by reference.

For example, the location of multidrug resistant (MDR) tumor cells, MDRHIV-infected cells or MDR infectious agents in a mammal having amultidrug resistant disease caused by a tumor or infectious agent can bedetermined by parenterally injecting the mammal with a polyspecificimmunoconjugate comprising (1) at least one antibody component thatbinds with a first epitope of a multidrug transporter protein, (2) atleast one antibody component that binds with a first epitope of anantigen that is associated with the tumor or infectious agent, and (3) adiagnostic agent. Subsequently, the mammal is injected with an antibodyor antibody fragment that binds with the polyspecific immunoconjugate inan amount that is sufficient to decrease the level of circulatingpolyspecific immunoconjugate by about 10-85% within 2 to 72 hours. Themammal is then scanned with a detector to locate the site or sites ofuptake of the polyspecific immunoconjugate. See Goldenberg, U.S. Pat.No. 4,624,846.

In an alternate approach, detection methods are improved by takingadvantage of the binding between avidin/streptavidin and biotin. Avidin,found in egg whites, has a very high binding affinity for biotin, whichis a B-complex vitamin. Streptavidin, isolated from Streptomycesavidinii, is similar to avidin, but has lower non-specific tissuebinding and therefore, streptavidin often is used in place of avidin. Abasic diagnostic method comprises administering an antibody compositeconjugated with avidin/streptavidin (or biotin), injecting a clearingcomposition comprising biotin (or avidin/streptavidin), andadministering a conjugate of a diagnostic agent and biotin (oravidin/streptavidin). Preferably, the biotin (or avidin/streptavidin)component of the clearing composition is coupled with a carbohydratemoiety (such as dextran) or a polyol group (e.g., polyethylene glycol)to decrease immunogenicity and permit repeated applications.

A modification of the basic method is performed by parenterallyinjecting a mammal with an antibody composite which has been conjugatedwith avidin/streptavidin (or biotin), injecting a clearing compositioncomprising biotin (or avidin/streptavidin), and parenterally injecting apolyspecific immunoconjugate according to the present invention, whichfurther comprises avidin/streptavidin (or biotin). See Goldenberg,international publication No. WO 94/04702, which is incorporated byreference.

In a further variation of this method, improved detection can beachieved by conjugating multiple avidin/streptavidin or biotin moietiesto a polymer which, in turn, is conjugated to an antibody component.Adapted to the present invention, antibody composites or polyspecificimmunoconjugates can be produced which contain multipleavidin/streptavidin or biotin moieties. Techniques for constructing andusing multiavidin/multistreptavidin and/or multibiotin polymerconjugates to obtain amplification of targeting are disclosed byGriffiths, international application No. PCT/US94/04295, which isincorporated by reference.

In another variation, improved detection is achieved by injecting atargeting antibody composite conjugated to biotin (oravidin/streptavidin), injecting at least one dose of anavidin/streptavidin (or biotin) clearing agent, and injecting adiagnostic composition comprising a conjugate of biotin (oravidin/streptavidin) and a naturally occurring metal atom chelatingprotein which is chelated with a metal detection agent. Suitabletargeting proteins according to the present invention would be ferritin,metallothioneins, ferredoxins, and the like. This approach is disclosedby Goldenberg et al., international application No. PCT/US94/05149,which is incorporated by reference.

Polyspecific immunoconjugates which comprise a radiolabel also can beused to detect multidrug resistant (MDR) tumor cells, MDR HIV-infectedcells or MDR infectious agents in the course of intraoperative andendoscopic examination using a small radiation detection probe. SeeGoldenberg U.S. Pat. No. 4,932,412, which is incorporated by reference.As an illustration of the basic approach, a surgical or endoscopysubject is injected parenterally with a polyspecific immunoconjugatecomprising (1) at least one antibody component that binds with a firstepitope of a multidrug transporter protein, (2) at least one antibodycomponent that binds with a first epitope of an antigen that isassociated with a tumor or infectious agent, and (3) a radioisotope.Subsequently, the surgically exposed or endoscopically accessed interiorof the body cavity of the subject is scanned at close range with aradiation detection probe to detect the sites of accretion of thepolyspecific immunoconjugate.

In a variation of this method, a photoactive agent or dye, such asdihematoporphyrin ether (Photofrin II), is injected systemically andsites of accretion of the agent or dye are detected by laser-inducedfluorescence and endoscopic imaging. See Goldenberg, internationalapplication No. PCT/US93/04098, which is incorporated by reference. Theprior art discloses imaging techniques using certain dyes that areaccreted by lesions, such as tumors, and which are in turn activated bya specific frequency of light. These methods are described, for example,in Dougherty et al., Cancer Res. 38: 2628 (1978); Dougherty, Photochem.Photobiol. 45: 879 (1987); Doiron et al. (eds.), PORPHYRIN LOCALIZATIONAND TREATMENT OF TUMORS (Alan Liss, 1984); and van den Bergh, Chem.Britain 22: 430 (1986), which are incorporated herein in their entiretyby reference.

In a basic technique, a subject is injected parenterally with apolyspecific immunoconjugate comprising (1) at least one antibodycomponent that binds with a first epitope of a multidrug transporterprotein, (2) at least one antibody component that binds with a firstepitope of an antigen that is associated with a tumor or infectiousagent, and (3) a photoactive agent or dye. Sites of accretion aredetected using a light source provided by an endoscope or during asurgical procedure.

The detection of polyspecific immunoconjugate during intraoperative orendoscopic examination can be enhanced through the use of secondantibody or avidin/streptavidin/biotin clearing agents, as discussedabove.

In these endoscopic techniques the detection means can be inserted intoa body cavity through an orifice, such as, the mouth, nose, ear, anus,vagina or incision. As used herein, the term “endoscope” is usedgenerically to refer to any scope introduced into a body cavity, e.g.,an anally introduced endoscope, an orally introduced bronchoscope, aurethrally introduced cystoscope, an abdominally introduced laparoscopeor the like. Certain of these may benefit greatly from further progressin miniaturization of components and their utility to practice themethod of the present invention will be enhanced as a function of thedevelopment of suitably microminiaturized components for this type ofinstrumentation. Highly miniaturized probes which could be introducedintravascularly, e.g., via catheters or the like, are also suitable foruse in the embodiments of the invention for localizing MDR tumor cells,MDR HIV-infected cells or MDR infectious agents.

7. Use of Polyspecific Immunoconjugates and Antibody Composites forTherapy

The present invention also contemplates the use of antibody compositesand polyspecific immunoconjugates for immunotherapy. An objective ofimmunotherapy is to deliver cytotoxic doses of radioactivity, toxin, ordrug to target.cells, while minimizing exposure to non-target tissues.The polyspecific immunoconjugates and antibody composites of the presentinvention are expected to have a greater binding specificity thanmultidrug transporter protein MAbs, since the polyspecificimmunoconjugates and antibody composites comprise moieties that bind toat least one multidrug transporter protein epitope and an antigenassociated with either a tumor or an infectious agent.

For example, a therapeutic polyspecific immunoconjugate may comprise anα-emitting radioisotope, a β-emitting radioisotope, a γ-emittingradioisotope, an Auger electron emitter, a neutron capturing agent thatemits α-particles or a radioisotope that decays by electron capture.Suitable radioisotopes include ¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y, ¹⁸⁶Re,¹⁸⁸Re, ⁶⁷Cu, ²¹¹At, and the like.

As discussed above, a radioisotope can be attached to an antibodycomposite directly or indirectly, via a chelating agent. For example,⁶⁷Cu, considered one of the more promising radioisotopes forradioimmunotherapy due to its 61.5 hour half-life and abundant supply ofbeta particles and gamma rays, can be conjugated to an antibodycomposite using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA). Chase,supra. Alternatively, ⁹⁰Y, which emits an energetic beta particle, canbe coupled to an antibody composite using diethylenetriaminepentaaceticacid (DTPA). Moreover, a method for the direct radiolabeling of theantibody composite with ¹³¹I is described by Stein et al., AntibodyImmunoconj. Radiopharm. 4: 703 (1991).

Alternatively, boron addends such as carboranes can be attached toantibody composites. Carboranes can be prepared with carboxyl functionson pendant side chains, as is well-known in the art. Attachment ofcarboranes to a carrier, such as aminodextran, can be achieved byactivation of the carboxyl groups of the carboranes and condensationwith amines on the carrier. The intermediate conjugate is thenconjugated to the antibody composite. After administration of thepolyspecific immunoconjugate, a boron addend is activated by thermalneutron irradiation and converted to radioactive atoms which decay byα-emission to produce highly toxic, short-range effects.

In addition, therapeutically useful polyspecific immunoconjugates can beprepared in which an antibody composite is conjugated to a toxin or achemotherapeutic drug. Illustrative of toxins which are suitablyemployed in the preparation of such conjugates are ricin, abrin, humanribonuclease, pokeweed antiviral protein, gelonin, diphtherin toxin, andPseudomonas endotoxin. See, for example, Pastan et al., Cell 47: 641(1986), and Goldenberg, CA—A Cancer Journal for Clinicians 44: 43(1994). Other suitable toxins are known to those of skill in the art.

Useful cancer chemotherapeutic drugs for the preparation of polyspecificimmunoconjugates include nitrogen mustards, alkyl sulfonates,nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purineanalogs, antibiotics, epipodophyllotoxins, platinum coordinationcomplexes, hormones, and the like. Chemotherapeutic drugs that areuseful for treatment of infectious agents include antiviral drugs (suchas AZT, 2′,3′-dideoxyinosine and 2′,3′-dideoxycytidine), antimalarialdrugs (such as chloroquine and its congeners, diaminopyrimidines,mefloquine), antibacterial agents, antifungal agents, antiprotozoalagents, and the like. Suitable chemotherapeutic agents are described inREMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (Mack Publishing Co.1990), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), which areincorporated by reference. Other suitable chemotherapeutic agents, suchas experimental drugs, are known to those of skill in the art.

In addition, therapeutically useful polyspecific immunoconjugates can beobtained by conjugating photoactive agents or dyes to an antibodycomposite. Fluorescent and other chromogens, or dyes, such as porphyrinssensitive to visible light, have been used to detect and to treatlesions by directing the suitable light to the lesion (cited above). Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy (Jori et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORSAND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain 22: 430 (1986)). Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy (Mew et al.,J. Immunol. 130: 1473 (1983); idem., Cancer Res. 45: 4380 (1985);Oseroff et al., Proc. Natl. Acad. Sci. USA 83: 8744 (1986); idem.,Photochem. Photobiol. 46: 83 (1987); Hasan et al., Prog. Clin. Biol.Res. 288: 471 (1989); Tatsuta et al., Lasers Surg. Med. 9: 422 (1989);Pelegrin et al., Cancer 67: 2529 (1991)—all incorporated in theirentirety herein by reference). However, these earlier studies did notinclude use of endoscopic therapy applications, especially with the useof antibody fragments or subfragments. Thus, the present inventioncontemplates the therapeutic use of polyspecific immunoconjugatescomprising photoactive agents or dyes. The general methodology isdescribed above in relation to the use of such polyspecificimmunoconjugates for diagnosis.

Moreover, therapeutically useful polyspecific immunoconjugates can beprepared in which an antibody composite is conjugated to a compound thatreverses multidrug resistance. Such “chemosensitizing agents” includeverapamil and its analogs, calmodulin antagonists, anthracycline andVinca alkaloid analogs, and the like. See, for example, Endicott et al.,Ann. Rev. Biochem. 58: 137 (1989), Ford et al., Pharmacol. Rev. 42: 155(1990) and Calabresi et al., PPO Updates 8: 1 (1994). See also Sarkadiet al., FASEB J. 8: 766 (1994), which provides methods to identifyhydrophobic peptide derivatives that reverse multidrug resistance. Thesepolyspecific immunoconjugates may be administered prior to, orconcurrent with, the administration of appropriate chemotherapeuticdrugs.

As an alternative, unconjugated chemosensitizing agents may beadministered with polyspecific immunoconjugates comprising a toxin orchemotherapeutic drug. Typical modes of administration and dosages ofchemosensitizing agents are described, for example, by Presant et al.,Am. J. Clin. Oncol. 9: 355 (1986), Cairo et al., Cancer Res. 49: 1063(1989), Miller et al., J. Clin. Oncol. 9: 37 (1991) and Calabresi etal., supra, Rubin, U.S. Pat. No. 5,005,588, and Levy, U.S. Pat. No.5,258,372, which are incorporated by reference.

In addition, therapeutic polyspecific immunoconjugates can comprise animmunomodulator moiety. As used herein, the term “immunomodulator”includes cytokines, stem cell growth factors, tumor necrosis factors(TNF) and hematopoietic factors, such as interleukins (e.g.,interleukin-1 (IL-1), IL-2, IL-3, IL-6 and IL-10), colony stimulatingfactors (e.g., granulocyte-colony stimulating factor (G-CSF) andgranulocyte macrophage-colony stimulating factor (GMC-SF)), interferons(e.g., interferons-α, -β and -γ), the stem cell growth factor designated“S1 factor,” erythropoietin and thrombopoietin. Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, interferon-′, TNF-α,and the like.

Such polyspecific immunoconjugates provide a means to deliver animmunomodulator to a target cell and are particularly useful againsttumor cells and mammalian cells that express an infectious agent antigenon the cell surface, such as HIV-infected cells. The cytotoxic effectsof immunomodulators are well known to those of skill in the art. See,for example, Klegerman et al., “Lymphokines and Monokines,” inBIOTECHNOLOGY AND PHARMACY, Pessuto et al. (eds.), pages 53-70 (Chapman& Hall 1993). As an illustration, type I interferons and interferon-γinduce an antiviral state in various cells by activating2′,5′-oligoadenylate synthetase and protein kinase. Moreover,interferons can inhibit cell proliferation by inducing increasedexpression of class I histocompatibility antigens on the surface ofvarious cells and thus, enhance the rate of destruction of cells bycytotoxic T lymphocytes. Furthermore, tumor necrosis factors, such asTNF-α, are believed to produce cytotoxic effects by inducing DNAfragmentation.

The present invention also contemplates two-, three- or four-steptargeting strategies to enhance antibody therapy. General techniquesinclude the use of antibody components conjugated with avidin,streptavidin or biotin, and the use of second antibodies that bind withthe primary immunoconjugate, as discussed above. See, for example,Goodwin et al., Eur. J. Nucl. Med. 9:209 (1984), Goldenberg et al., J.Nucl. Med. 28:1604 (1987), Hnatowich et al., J. Nucl. Med. 28: 1294(1987), Paganelli et al., Cancer Res. 51: 5960 (1991), Goldenberg,international publication No. WO 92/19273, Sharkey. et al., Int. J.Cancer 51: 266 (1992), and Goldenberg, international application No. WO94/04702, which are incorporated by reference. Also, see Griffiths,international application No. PCT/US94/04295, which describes a methodusing multiavidin and/or multibiotin polymer conjugates, and Goldenberget al., international application No. PCT/US94/05149, which disclosesimproved methods for therapy with chelatable radiometals.

For example, a mammal having a multidrug resistant disease caused by atumor or infectious agent may be treated by parenterally injecting themammal with a polyspecific immunoconjugate comprising (1) at least oneantibody component that binds with a first epitope of a multidrugtransporter protein, (2) at least one antibody component that binds witha first epitope of an antigen that is associated with the tumor orinfectious agent, and (3) a therapeutic agent. Subsequently, the mammalis injected with an antibody or antibody fragment that binds with thepolyspecific immunoconjugate in an amount that is sufficient to decreasethe level of circulating polyspecific immunoconjugate by about 10-85%within 2 to 72 hours.

In an alternative approach to enhancing the therapeutic index comprisesadministering an antibody composite conjugated with avidin/streptavidin(or biotin), injecting a clearing composition comprising biotin (oravidin/streptavidin), and administering a conjugate of a therapeuticagent and avidin/streptavidin (or biotin), as discussed above.

The present invention also contemplates a method of therapy usingunconjugated antibody composites. Investigators have found thatP-glycoprotein antibodies can restore drug sensitivity in multidrugresistant cultured cells and multidrug resistant human tumor xenograftsin nude mice. Grauer et al., European patent application No. EP-0 569141, Rittmann-Grauer et al., Cancer Res. 52: 1810 (1992), Pearson etal., J. Nat'l Cancer Inst. 83: 1386 (1991), and Iwahashi et al., CancerRes. 53: 5475 (1993). P-glycoprotein antibodies also can inhibit thegrowth of multidrug resistant human xenografts in nude mice. Grauer etal., European patent application No. EP-0 569,141. Accordingly, the morespecific antibody composites of the present invention provide animproved method to treat a mammal having a multidrug resistant diseasecaused by a tumor or infectious agent in which the multidrug resistantcells overexpress P-glycoprotein. Moreover, the antibody composites ofthe present invention can be used to inhibit active drug efflux ininfectious agents and thus, restore sensitivity to chemotherapy.

Antibody composites may be administered alone, or in conjugation withthe conventional chemotherapeutic agents described above. Modes ofchemotherapeutic administration and suitable dosages are well known tothose of skill in the art. See, for example, REMINGTON'S PHARMACEUTICALSCIENCES, 18th Ed. (Mack Publishing Co. 1990), and GOODMAN AND GILMAN'STHE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan PublishingCo. 1985).

In general, the dosage of administered polyspecific immunoconjugates andantibody composites will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history. Typically, it is desirable to provide therecipient with a dosage of polyspecific immunoconjugate or antibodycomposite which is in the range of from about 1 pg/kg to 10 mg/kg(amount of agent/body weight of patient), although a lower or higherdosage also may be administered as circumstances dictate.

Administration of polyspecific immunoconjugates or antibody compositesto a patient can be intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, intrapleural, intrathecal, by perfusionthrough a regional catheter, or by direct intralesional injection. Whenadministering polyspecific immunoconjugates or antibody composites byinjection, the administration may be by continuous infusion or by singleor multiple boluses.

Polyspecific immunoconjugates having a boron addend-loaded carrier forthermal neutron activation therapy will normally be effected in similarways. However, it will be advantageous to wait until non-targetedpolyspecific immunoconjugate clears before neutron irradiation isperformed. Clearance can be accelerated using an antibody that binds tothe polyspecific immunoconjugate. See U.S. Pat. No. 4,624,846 for adescription of this general principle.

The polyspecific immunoconjugates and antibody composites of the presentinvention can be formulated according to known methods to preparepharmaceutically useful compositions, whereby polyspecificimmunoconjugates or antibody composites are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990).

For purposes of therapy, a polyspecific immunoconjugate (or antibodycomposite) and a pharmaceutically acceptable carrier are administered toa patient in a therapeutically effective amount. A combination of apolyspecific immunoconjugate (or antibody composite) and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. In the present context, an agent is physiologicallysignificant if its presence results in the inhibition of the growth oftarget cells, or in the increased susceptibility of target cells to achemotherapeutic agent.

Additional pharmaceutical methods may be employed to control theduration of action of a polyspecific immunoconjugate or antibodycomposite in a therapeutic application. Control release preparations canbe prepared through the use of polymers to complex or adsorb thepolyspecific immunoconjugate or antibody composite. For example,biocompatible polymers include matrices of poly(ethylene-co-vinylacetate) and matrices of a polyanhydride copolymer of a stearic aciddimer and sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992).The rate of release of a polyspecific immunoconjugate (or antibodycomposite) from such a matrix depends upon the molecular weight of thepolyspecific immunoconjugate (or antibody composite), the amount ofpolyspecific immunoconjugate (or antibody composite) within the matrix,and the size of dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed. (1990).

The present invention also contemplates a method of treatment in whichimmunomodulators are administered to prevent, mitigate or reverseradiation-induced or drug-induced toxicity of normal cells, andespecially hematopoietic cells. Adjunct immunomodulator therapy allowsthe administration of higher doses of cytotoxic agents due to increasedtolerance of the recipient mammal. Moreover, adjunct immunomodulatortherapy can prevent, palliate, or reverse dose-limiting marrow toxicity.Examples of suitable immunomodulators for adjunct therapy include G-CSF,GM-CSF, thrombopoietin, IL-1, IL-3, and the like. The method of adjunctimmunomodulator therapy is disclosed by Goldenberg, U.S. Pat. No.5,120,525, which is incorporated by reference.

Those of skill in the art are aware that an antibody component is justone example of a moiety that can be used to target particular cells.Other useful targeting moieties include non-antibody proteins, peptides,polypeptides, glycoproteins, lipoproteins, or the like, e.g., growthfactors, enzymes, receptor proteins, immunomodulators and hormones. Forexample, Sarkadi et al., The FASEB Journal 8: 766 (1994) ; which isincorporated by reference, provides methods for identifying hydrophobicpeptides that interact with P-glycoprotein. As an illustration, apolyspecific conjugate suitable for diagnosis and/or treatment ofcertain multidrug resistant breast cancers would comprise a hydrophobicpeptide that binds with P-glycoprotein, an epidermal growth factormoiety that binds with the c-erb B2 proto-oncogene product, and adiagnostic or therapeutic agent.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLE 1 Production of Antibody Components: Murine P-Glycoprotein MAband Anti-CEA MAb

1. Production of Monoclonal Antibodies

Methods for producing anti-P-glycoprotein murine monoclonal antibodiesare well-known to those of skill in the art, as discussed above. Oneapproach is to immunize mice with cells, or cellular membranes, thatcontain an abundance of P-glycoprotein. Cells that over-expressP-glycoprotein can be obtained by selecting and enriching cells thatexpress the MDR phenotype from a human continuous cell line. See, forexample, Gottesman, “Drug-Resistant Mutants: Selection and DominanceAnalysis,” in METHODS IN ENZYMOLOGY, VOL. 151, Colowick et al., (eds.),pages 113-121 (Academic Press 1987), and Clynes et al., Cytotechnology12: 231 (1993).

A general method for using MDR cells to produce anti-P-glycoproteinmonoclonal antibodies is described, for example, by Rittmann-Grauer etal., Cancer Res. 52: 1810 (1992), which is incorporated by reference.Briefly, six week old female BALB/c mice are injected intraperitoneally(i.p.) with 5×10⁶ MDR cells that have been scraped from the surface oftissue culture flasks. Three weeks later, mice receive a second i.p.injection of 5×10⁶ MDR cells. Four days prior to fusion, mice receive afinal intravenous boost of 5×10⁶ MDR cells. Splenocytes from theimmunized mice are fused with murine myeloma cells, SP2/0-Ag 14,according to the method of Gerhard, “Fusion of Cells in Suspension andOutgrowth of Hybrids in Conditioned Medium,” in MONOCLONAL ANTIBODIES,Kennet et al. (eds.), pages 370-371 (Plenum Publishing Corp. 1981).

Anti-P-glycoprotein hybridoma cultures are initially screened using anindirect ELISA with a horseradish peroxidase conjugate of goatanti-mouse immunoglobulin. Monolayers of the MDR cells and thedrug-sensitive parental cell line are cultured in 96-well microtiterplates. Cells are fixed with 0.01% glutaraldehyde for 45 minutes at roomtemperature, the fixative is removed, cells are washed three times withphosphate-buffered saline (PBS), and the microtiter wells are blockedwith 10% bovine serum albumin for at least 45 minutes. Fifty microlitersof hybridoma supernatants are added to the microtiter wells and allowedto incubate for one hour at 37° C. Plates are then washed with PBS andincubated with 50 μl of peroxidase-conjugated goat anti-mouseimmunoglobulin diluted 1:1000 in PBS with 10% horse serum. Followingfive washes with PBS, positive clones are identified by the addition of100 μl of a solution containing 1 mg/ml O-phenylenediamine, 0.1%hydrogen peroxide, 50 mM citrate, and 100 mM sodium phosphate buffer (pH5.0). The reaction is quenched by the addition of 50 μl 4N sulfuricacid, and the plates are read at 490 nm.

Clones that produce a five-fold or greater ELISA signal for the MDRcells, compared with drug-sensitive cells, are expanded. Hybridoma cellsthat produce anti-P-glycoprotein antibodies are injected into BALB/cmice for ascites production according to the procedure of Hoogenraad et.al., J. Immunol. Methods 61: 317 (1983). Anti-P-glycoprotein antibodiesare purified from the ascites fluid using protein A chromatography. See,for example, Langone et al., J. Immunol. Methods 51: 3 (1982).

The production of highly specific anti-CEA MAb, has been described byHansen et al., Cancer 71: 3478 (1993), which is incorporated byreference. Briefly, a 20 gram BALB/c female mouse was immunizedsubcutaneously with 7.5 μg of partially-purified CEA in complete Freundadjuvant. On day 3, the mouse was boosted subcutaneously with 7.5 μg ofCEA in incomplete Freund adjuvant and then, the mouse was boostedintravenously with 7.5 μg of CEA in saline on days 6 and 9. On day 278,the mouse was given 65 μg of CEA intravenously in saline and 90 μg ofCEA in saline on day 404. On day 407, the mouse was sacrificed, a cellsuspension of the spleen was prepared, the spleen cells were fused withmurine myeloma cells, SP2/0-Ag 14 (ATCC CRL 1581) using polyethyleneglycol, and the cells were cultured in medium containing 8-azaguanine.Hybridoma supernatants were screened for CEA-reactive antibody using an¹²⁵I-CEA radioimmunoassay (Roche; Nutley, N.J.). Positive clones wererecloned.

2. The Production of Antibody Fragments

As described above, proteolysis provides one method for preparingantibody fragments. This technique is well-known to those of skill inthe art. For example, see Coligan et al., supra, at pp. 2.8.1-2.8.10.Also see Stanworth et al. “Immunochemical Analysis of Human and RabbitImmunoglobulins and Their Subunits,” in HANDBOOK OF EXPERIMENTALIMMUNOLOGY, Vol. 1, Weir (ed.), pages 12.1-12.46 (Blackwell Scientific1986), and Parham, “Preparation and Purification of Active Fragmentsfrom Mouse Monoclonal Antibodies,” Id. at pages 14.1-14.23.

As an example, preactivated papain can be used to prepare F(ab)₂fragments from IgG1 or Fab fragments from IgG2a and IgG2b, as follows.Papain is activated by incubating 2 mg/ml papain (2×recrystallizedsuspension, Sigma #P3125) and 0.05 M cysteine (free-base, crystalline;Sigma #C7755) for 30 minutes in a 37° C. water bath. To remove cysteine,the papain/cysteine mixture is applied to a PD-10 column (Pharmacia#G-25), which has been equilibrated with 20 ml of acetate/EDTA buffer(0.1 M acetate with 3 mM EDTA, pH 5.5). Fractions are assayed bymeasuring absorbance at 280 nm, and the two or three fractions thatcontain protein are pooled. The concentration of preactivated papain isdetermined by using the formula: (absorbance at 280 nm)/2.5=mg.preactivated papain/ml.

To prepare antibody for digestion, 10 mg of antibody in 2 to 5 ml of PBSare dialyzed against acetate/EDTA buffer. Five hundred micrograms ofpreactivated papain are added to the dialyzed antibody solution, and themixture is vortexed. After a 6-12 hour incubation in a 37° C. waterbath, papain is inactivated by adding crystalline iodoacetamide (Sigma#I6125) to a final concentration of 0.03 M. The mixture is then dialyzedagainst 1 liter of PBS (pH 8.0) at 4° C. for 6-12 hours.

To remove undigested antibody and Fc fragments, the mixture is appliedto a protein A-Sepharose column which has been equilibrated in PBS (pH8.0). Unbound fractions are collected in 2 ml aliquots and pooled. Afterconcentrating the pool to a total volume of 5 ml or less, protein isfractionated by size-exclusion chromatography and the results areanalyzed by SDS-PAGE.

EXAMPLE 2 Preparation of Antibody Composite:Anti-P-Glycoprotein/anti-CEA Bispecific Antibody

A bispecific F(ab′)₂ antibody composite is prepared from an Fab′fragment of an anti-P-glycoprotein monoclonal antibody and an Fab′fragment of a monoclonal antibody specific for CEA, using the methodsdescribed above. Also, see Goldenberg, international publication No. WO92/19273, which is incorporated by reference. Briefly, the interchaindisulfide bridges are reduced carefully with cysteine, taking care toavoid light-heavy chain cleavage, to form Fab′-SH fragments. The SHgroup(s) of one antibody fragment is(are) activated with an excess ofbis-maleimide linker (1,1′-(methylenedi-1,4-phenylene)bismaleimide(Aldrich Chemical Co.; Milwaukee, Wis.). The second antibody fragment isalso converted to Fab′-SH and then reacted with the activated firstantibody fragment to obtain a bispecific antibody composite.

EXAMPLE 3 Preparation of Polyspecific Immunoconjugate

A polyspecific immunoconjugate can be prepared by binding a therapeuticor diagnostic agent to the bispecific antibody composite, described inExample 2. As an example, the anti-P-glycoprotein/anti-CEA composite canbe conjugated with doxorubicin via dextran, using the method of Shih etal., Int. J. Cancer 41:832-839 (1988). Briefly, amino dextran isprepared by dissolving one gram of dextran (m.w. 18 kD; Sigma ChemicalCo.; St. Louis, Mo.) in 70 ml of water. The dextran is partiallyoxidized to form polyaldehyde dextran by adding 0.5 gram of sodiummetaperiodate, and stirring the solution at room temperature overnight.After concentrating the mixture with an Amicon cell (YM10 membrane;MWCO=10,000), the polyaldehyde dextran is purified by Sephadex G-25chromatography and lyophilized to give about 900 grams of white powder.Polyaldehyde dextran is then treated with two equivalents of1,3-diamino-2-hydroxypropane in aqueous phase for 24 hours at roomtemperature. The resultant Schiff base is stabilized by addition ofsodium borohydride (0.311 mmol per 2.15 mmol of1,3-diamino-2-hydroxypropane) to the mixture. The mixture is allowed toincubate at room temperature for six hours. Amino dextran is purifiedusing a Sephadex G-25 column.

Doxorubicin (Sigma Chemical Co.; St. Louis, Mo.) is activated by addingone milliliter of anhydrous DMF to 0.1 mmole of doxorubicin in a driedReacti-vial, followed by a solution of N-hydroxysuccinimide (23 mg, 0.2mmole; Sigma) in 750 μl of anhydrous DMF and a solution of1,3-dicyclohexylcarbodiimide (41.5 mg, 0.2 mmol; Sigma) in 750 μl ofanhydrous DMF. The reaction mixture is stirred in the dark at roomtemperature for 16 hours under anhydrous conditions. The precipitate isthen centrifuged and the solution is stored in a sealed bottle at −20°C.

Doxorubicin-dextran intermediate conjugate is prepared by dissolvingaminodextran (18 kD; 10 mg) in two milliliters of PBS (pH 7.2) andgradually adding 0.7 ml of the above N-hydroxy-succinimide-activateddoxorubicin solution. Thus, 50 moles of doxorubicin are present per moleof aminodextran. The solution is stirred at room temperature for fivehours and after removing any precipitate, the conjugate is purifiedusing a Sephadex G-25 column. Doxorubicin-dextran conjugate is typicallycharacterized by a doxorubicin/dextran ratio of 14.

Alternatively, doxorubicin-dextran conjugate is prepared by reactingdoxorubicin with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, asdescribed by Shih et al., Int. J. Cancer 41:832-839 (1988). Also, seeShih et al., Cancer Research 51:4192-4198 (1991).

The bispecific antibody conjugate (25 mg) in 5 ml of PBS (pH 5.5) isoxidized in the dark by treatment with sodium metaperiodate (800 μl of a21.5 mg/ml solution) at room temperature for 60 minutes. The reactionmixture is then treated with ethylene glycol (50 μl) to decompose theunreacted periodate and the oxidized antibody fragment is purified usinga Sephadex G-25 column equilibrated in 0.05 M HEPES (pH 7.4).Subsequently, the oxidized fragment is concentrated to 5 mg/ml in 0.05 MHEPES (pH 7.4) and reacted with the doxorubicin-dextran conjugate (22mg). After 24 hours at room temperature, the Schiff base is reduced byNaBH₃CN. Conjugated antibody is purified using a Sepharose CL-6B column.

EXAMPLE 4 Preparation of an Polyspecific Immunoconjugate Comprising aRadioisotope

A polyspecific immunoconjugate can be prepared in which a radioisotopeis bound to one or more antibody components via a chelator. As anillustration, the antibody composite of Example 2 may be conjugated witheither aminobenzyl diethylenetriaminepentaacetic acid (DTPA) or aderivative of DTPA containing the long-chain linker, —CSNH(CH₂)₁₀NH₂(LC-DTPA). Briefly, the antibody composite (2.5 mg in about onemilliliter of 50 mM acetate-buffered 0.9% saline [ABS; pH 5.3]) isoxidized in the dark by treatment with sodium metaperiodate (210 μl of a5.68 mg/ml solution) at 0° C. for one hour. The reaction mixture istreated with ethylene glycol (20 μl) to decompose the unreactedperiodate and the oxidized antibody fragment is purified using aSephadex G-50/80 column (Pharmacia; Piscataway, N.J.) equilibrated inPBS (pH 6.1). The oxidized fragment is then reacted with excess DTPA orLC-DTPA. After 40 hours at room temperature, the Schiff base is reducedby NaBH₃CN. Conjugated antibody composite is then purified using acentrifuged size-exclusion column (Sephadex G-50/80) equilibrated in 0.1M acetate (pH 6.5). The concentrations of antibody conjugates aredetermined by measuring absorbance at 280 nm.

The ratio of chelator molecules per molecule of antibody composite isdetermined by a metal-binding assay. The assay is performed by mixing analiquot of the antibody conjugate with 0.1 M ammonium acetate (pH 7) and2 M triethanolamine, and incubating the mixture at room temperature witha known excess of cobalt acetate spiked with ⁵⁷cobalt acetate. After 30minutes, EDTA (pH 7) is added to a final concentration of 10 mM. After afurther 10 minute incubation, the mixture is analyzed by instant thinlayer chromatography (ITLC) using 10 mM EDTA for development. Thefraction of radioactivity bound to antibody is determined by countingsections of ITLC strips on a gamma counter. Typically, the results willshow that there are about 6 molecules of DTPA per antibody component andabout 5 molecules of LC-DTPA per antibody component.

Antibody conjugates are labeled with ⁹⁰yttrium, as follows. Briefly,commercially available ⁹⁰yttrium chloride (DuPont NEN; 17.68 μl; 5.63mCi) is buffered with 35.4 μl of 0.5 M acetate (pH 6.0). The solution isallowed to stand for 5-10 minutes at room temperature, and then used forradiolabeling.

⁹⁰Yttrium-labeled antibody composite-DTPA is prepared by mixing⁹⁰yttrium acetate (128.7 μCi) with antibody composite-DTPA (30 μg; 8.3μl), incubating at room temperature for one hour, and diluting with 90μl of 0.1 M acetate (pH 6.5). ⁹⁰Yttrium-labeled antibodycomposite-LC-DTPA is prepared by mixing yttrium acetate (109.5 μci) withantibody composite-LC-DTPA (30 μg; 7.6 μl), incubating at roomtemperature for one hour, and diluting with 90 μl of 0.1 M acetate (pH6.5).

The extent of ⁹⁰yttrium incorporation can be analyzed by incubating thelabeling mixture with 10 mM EDTA for ten minutes, followed by ITLCexamination using 10 mM EDTA for development. In this assay, unboundyttrium migrates with the solvent front, while antibody-bound yttriumremains at the origin. The presence of any colloidal ⁹⁰yttrium isassayed by ITLC (co-spotted with human serum albumin) using awater:ethanol:ammonia (5:2:1) solution for development. In this system,the fraction of radioactivity at the origin represents colloidal⁹⁰yttrium. In addition, all labeling mixtures may be analyzed usingradio-high pressure liquid chromatography. Typically, 90 to 96% of⁹⁰Yttrium is incorporated into the resultant polyspecificimmunoconjugate.

EXAMPLE 5 Treatment of Colon Cancer with ⁹⁰Yttrium-Labeled PolyspecificImmunoconjugate and G-CSF

A patient has a carcinoembryonic antigen (CEA) blood titer of 55 ng/mldue to peritoneal spread of a colon cancer which had been resected twoyears earlier and found to be a Dukes' C lesion. Since previouschemotherapy with fluorouracil had been unsuccessful, the patientpresents for experimental therapy. The patient is given a 35 mCi dose ofthe ⁹⁰ttrium-labeled polyspecific immmunoconjugate prepared in Example4, by intraperitoneal injection. Two days later, an infusion of 5 μg/kgG-CSF (such as NEUPOGEN [Amgen, Inc.; Thousand Oaks, Calif.]) isinstituted intravenously, and the patient's hematologic values aremonitored thereafter. No significant drop in white blood cell count isnoted, thus permitting a repetition of the radioimmunotherapy threeweeks later, followed again by G-CSF therapy. A third treatment is giventwo months later, and radiological evidence of some tumor and ascitesreduction is noted four weeks later. Thus, the patient is able totolerate higher and more frequent doses of the radioimmunotherapy agent.

EXAMPLE 6 Preparation of an Antibody Composite Targeted to MultidrugResistant Psuedomonas Aeruginosa

Those of skill in the art can use standard methods to produce antibodiesagainst a multidrug transporter protein of an infectious agent. As anillustration, a bispecific antibody can be constructed which is targetedto multidrug resistant Psuedomonas aeruginosa. Antibody components thatbind to OprK, a multidrug transporter protein of Psuedomonas aeruginosa,can be obtained using OprK protein that is overexpressed by bacterialcells. For example, the OprK gene can be synthesized using mutuallypriming long oligonucleotides which are based upon the nucleotidesequence disclosed in Poole et al., J. Bacteriol. 175: 7363 (1993). See,for example, Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULARBIOLOGY, pages 8.2.8 to 8.2.13 (Wiley Interscience 1990). Also, seeWosnick et al., Gene 60:115 (1987). Moreover, current techniques usingthe polymerase chain reaction provide the ability to synthesize genes aslarge as 1.8 kilobases in length. Adang et al., Plant Molec. Biol.21:1131 (1993); Bambot et al., PCR Methods and Applications 2:266(1993).

The OprK gene is then cloned into a prokaryotic expression vector whichis subsequently introduced into competent E. coli cells, using standardtechniques. See, for example, Ausubel et al., supra, at pages16.1.1-16.7.8. OprK protein is isolated from the host cells usingstandard techniques. (Id.)

Alternatively, OprK protein can be isolated from Psuedomonas aeruginosaewhich have been selected for the multidrug resistant phenotype, asdescribed by Poole et al., supra.

Isolated OprK protein is used to generate anti-OprK MAb, as describedabove. Also, see Mole et al., “Production of Monoclonal AntibodiesAgainst Fusion Proteins Produced in Eschericia coli,” in DNA CLONING,VOLUME III: A PRACTICAL APPROACH, Glover (ed.), pages 113-139 (IRL Press1987), and Dean “Preparation and Testing of Monoclonal Antibodies toRecombinant Proteins,” in METHODS IN MOLECULAR BIOLOGY, VOLUME 10:IMMUNOCHEMICAL PROTOCOLS, Manson (ed.) pages 43-63 (The Humana Press,Inc. 1992).

Thus, anti-OprK MAb, or a fragment thereof, provides one antibodycomponent of a bispecific antibody. The second antibody component, whichbinds with a different antigen associated with the exterior surface ofPsuedomonas aeruginosa, may be obtained using the general techniquesdescribed above. Alternatively, suitable monoclonal antibodies can bepurchased from American Type Culture Collection (Rockville, Md.), suchas antibodies against Psuedomonas aeruginosa lipopolysaccharide (ATCCCRL Nos. 8753, 8754, 8795, 8796 and 8797), lipoprotein H2 of the outerenvelop of Psuedomonas aeruginosa (ATCC CRL 1783), Psuedomonasaeruginosa type a flagella (ATCC HB 9130), and Psuedomonas aeruginosatype b flagella (ATCC HB 9129).

An antibody composite comprising a moiety that binds OprK and a moietythat binds an exterior surface antigen of P. aeruginosa can be preparedusing the methods described in Example 2.

EXAMPLE 7 Preparation and Use of an ¹¹¹Indium-Labeled PolyspecificImmunoconjugate Targeted to Multidrug Resistant Psuedomonas Aeruginosa

Antibody composite-chelator conjugates are prepared as described inExample 4. The conjugates are labeled with ¹¹¹Indium, as follows.Briefly, ¹¹¹Indium chloride is buffered at pH 5.5 using ammonium acetatesuch that the final acetate concentration is about 0.2 M. ¹¹¹Indiumacetate is added to a solution of the antibody composite-chelatorconjugate in 0.1 M acetate (pH 6.5), and the mixture is incubated forabout one hour. Typically, reaction mixtures contain either 10 μg ofantibody composite-DTPA and 73 μCi of ¹¹¹Indium, or 10 μg of antibodycomposite-LC-DTPA and 126.7 μCi of ¹¹¹Indium. The extent of ¹¹¹Indiumincorporation is analyzed using ITLC, as described above.

A patient with granulocytopenia has Pseudomonas aeruginosa pneumoniawhich is no longer responsive to carbenicillin treatment. Fourmillicuries of ¹¹¹Indium-labeled polyspecific immmunoconjugate areinjected intravenously and after waiting at least 24 hours, the patientis scanned with a gamma camera. Foci of increased radioactivity appearas nodes in the lower lobes of the lung, indicating the presence ofpneumonic infiltrates with multidrug resistant Pseudomonas aeruginosa. Acourse of therapy is designed in which an aminoglycoside andcarbenicillin are administered with nonradioactive polyspecificimmunoconjugate that comprises an OprK-binding moiety, a moiety thatbinds an exterior surface antigen of Pseudomonas aeruginosa and achemosensitizing agent.

EXAMPLE 8 Preparation of a ^(99m)Tc-Labeled Polyspecific ImmunoconjugateTargeted to Multidrug Resistant Psuedomonas Aeruginosa

An antibody composite is prepared which binds OprK and E87 antigen, anexterior surface antigen of Psuedomonas aeruginosa. General techniquesfor preparing the antibody composite are described in Example 6, andpreparation of anti-E87 monoclonal antibodies is described by Sawada etal., U.S. Pat. No. 5,089,262.

The antibody composite is labeled with ^(99m)Tc using methods that arewell-known to those of skill in the art. See, for example, Crockford etal., U.S. Pat. No. 4,424,200, Paik et al., U.S. Pat. No. 4,652,440,Baidoo et al., Cancer Research (Suppl.) 50: 799s (1990), Griffiths etal., Cancer Research 51: 4594 (1991), Pak et al., U.S. Pat. No.5,053,493, Griffiths et al., U.S. Pat. No. 5,128,119, Lever et al., U.S.Pat. No. 5,095,111, and Dean et al., U.S. Pat. No. 5,180,816.

As an illustration, ^(99m)Tc-labeled polyspecific immunoconjugate can beobtained as described by Hansen et al., U.S. Pat. No. 5,328,679.Briefly, a solution of 0.075 M SnCl₂ (solution I) is prepared bydissolving 3350 mg SnCl·2H₂ 0 in one milliliter of 6 N HCl and dilutingthe resultant solution with sterile H₂ 0 which has been purged withargon. A solution of 0.1 M NaK tartrate in 0.05 M NaAc (pH 5.5)[solution II] is prepared with sterile H₂ 0 purged with argon. onevolume of solution I is mixed with 26 volumes of solution II, and theresultant solution III is filter sterilized and purged with argon.

A solution of antibody composite is reduced with 20 mM cysteine, andexcess cysteine is removed by gel filtration. The reduced antibodycomposite (2 mg/ml) is stabilized at pH 4.5 in 0.05 M NaOAc buffercontaining 0.15 M saline. The resultant solution IV is filter sterilizedand purged with argon. Solution IV is mixed with a sufficient amount ofsolution III to obtain a final concentration of 123 μg Sn per mg ofreduced antibody composite. The resultant solution V is adjusted to a pHof 4.5-4.8.

A sterile solution of sodium pertechnetate (10 mCi) in saline is addedto an aliquot of solution V which contains 1.25 mg antibody compositeand stable stannous ions, and the mixture is gently agitated. Labelingis quantitative within 5 minutes. The resultant solution of^(99m)Tc-labeled polyspecific immunoconjugate is ready for immediateinjection.

The ^(99m)Tc-labeled polyspecific immunoconjugate is administered to asubject, and sites of infection caused by multidrug resistantPsuedomonas aeruginosa are localized using single-photon emissioncomputed tomography.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those in the art to which theinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference in its entirety.

What is claimed is:
 1. A method for treating a mammal having either amultidrug resistant tumor that expresses a tumor associated antigen or amultidrug resistant disease caused by an infectious agent, said methodcomprising the step of: (1) administering an antibody composite to themammal, wherein said antibody composite comprises: (a) at least oneantibody component that binds with a first epitope of a multidrugtransporter protein, (b) at least one antibody component that binds witha first epitope of an antigen, wherein said antigen is associated withsaid tumor or said infectious agent, and either (c) at least onetherapeutic agent, thereby forming a polyspecific immunoconjugate, or(c′) a biotin-binding molecule or biotin for subsequent binding to abiotin-linked or biotin-binding molecule-linked therapeutic agent. 2.The method of claim 1, wherein said antibody components are selectedfrom the group consisting of: (a) a murine monoclonal antibody; (b) ahumanized antibody derived from (a); (c) a human monoclonal antibody;(d) a subhuman primate antibody; and (e) an antibody fragment derivedfrom (a), (b), (c) or (d).
 3. The method of claim 2, wherein saidantibody fragment is selected from the group consisting of F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, sFv and minimal recognition unit.
 4. The methodof claim 1, wherein said multidrug transporter protein is selected fromthe group consisting of P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA,NorA, QacA, CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC,MsrA, DrrA, DrrB, TlrC, Bmr, TetA and OprK.
 5. The method of claim 4,wherein said therapeutic agent of said polyspecific immunoconjugate isselected from the group consisting of radioisotope, boron addend, toxin,immunomodulator, photoactive agent or dye, cancer chemotherapeutic drug,antiviral drug, antifungal drug, antibacterial drug, antiprotozoal drugand a chemosensitizing agent.
 6. The method of claim 5, wherein saidradioisotope is selected from the group consisting of α-emitters,β-emitters, Auger electron emitters, neutron capturing agents that emitα-particles and radioisotopes that decay by electron capture.
 7. Themethod of claim 6, wherein said radioisotope is selected from the groupconsisting of ¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu and ²¹¹At.8. The method of claim 5, further comprising the step of administering achemosensitizing agent to said mammal.
 9. The method of claim 5, whereinsaid therapeutic agent is a chemosensitizing agent.
 10. The method ofclaim 9, further comprising the step of administering a chemotherapeuticagent selected from the group consisting of cancer chemotherapeuticdrug, antibacterial drug, antiviral drug, antifungal drug andantiprotozoal drug.
 11. The method of claim 5, further comprising thestep of administering an immunomodulator, wherein said immunomodulatoris selected from the group consisting of cytokine, stem cell growthfactor and hematopoietic factor.
 12. The method of claim 11, whereinsaid cytokine is granulocyte-colony stimulating factor.
 13. The methodof claim 11, wherein said hematopoietic factor is thrombopoietin. 14.The method of claim 11, wherein said immunomodulator is administeredprior to or simultaneously with said administration of said polyspecificimmunoconjugate.
 15. The method of claim 11, wherein saidimmunomodulator is administered subsequent to said administration ofsaid polyspecific immunoconjugate.
 16. The method of claim 5, whereinsaid therapeutic agent is a photoactive agent or dye, and wherein saidmethod further comprises the steps of: (2) surgically exposing orendoscopically accessing the interior of the body cavity of saidsubject; and (3) treating sites of accretion of said polyspecificimmunoconjugate to light, wherein said treatment activates saidphotoactive agent or dye.
 17. The method of claim 4, wherein saidantibody composite comprises a biotin-binding molecule or biotin, andwherein said method further comprises the steps of: (2) parenterallyinjecting a clearing composition comprised of: (i) biotin, when saidantibody composite is conjugated with a biotin-binding molecule, or (ii)a biotin-binding, molecule, when said antibody composite is conjugatedwith biotin, and allowing said clearing composition to substantiallyclear said antibody composite from sites that do not contain multidrugresistant (MDR) cells or MDR infectious agents; and (3) parenterallyinjecting a therapeutic composition comprised of: (i) biotin, when saidantibody composite is conjugated with a biotin-binding molecule, or (ii)a biotin-binding molecule, when said antibody composition is conjugatedwith biotin, and a therapeutic agent which is linked to said biotin orsaid biotin-binding molecule.
 18. The method of claim 17, wherein saidbiotin-binding molecule is avidin or streptavidin.
 19. The method ofclaim 18, wherein said therapeutic agent is selected from the groupconsisting of radioisotope, boron addend, toxin, immunomodulator,photoactive agent or dye, cancer chemotherapeutic drug, antiviral drug,antifungal drug, antibacterial drug, antiprotozoal drug and achemosensitizing agent.
 20. The method of claim 19, wherein saidradioisotope is selected from the group consisting of α-emitters,β-emitters, Auger electron emitters, neutron capturing agents that emitα-particles and radioisotopes that decay by electron capture.
 21. Themethod of claim 20, wherein said radioisotope is selected from the groupconsisting of ¹⁹⁸Au, ³²P , ¹²⁵I, ¹³¹I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu and²¹¹At.
 22. The method of claim 1, wherein said antibody composite is apolyspecific immunoconjugate, and wherein said method further comprisesthe stop of: (2) parenterally injecting said mammal with an antibody orantibody fragment that binds with said polyspecific immunoconjugate inan amount that is sufficient to decrease the level of circulatingpolyspecific immunoconjugate by about 10-85% within 2 to 72 hours. 23.The method of claim 22, wherein said antibody components are selectedfrom the group consisting of: (a) a murine monoclonal antibody; (b) ahumanized antibody derived from (a); (c) a human monoclonal antibody;(d) a subhuman primate antibody; and (e) an antibody fragment derivedfrom (a), (b), (c) or (d).
 24. The method of claim 23, wherein saidantibody fragment is selected from the group consisting of F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, sFv and minimal recognition unit.
 25. The methodof claim 24, wherein said multidrug transporter protein is selected fromthe group consisting of P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA,NorA, QacA, CMIA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC,MsrA, DrrA, DrrB, TlrC, Bmr, TetA and OprK.
 26. The method of claim 24,wherein said therapeutic agent is selected from the group consisting ofradioisotope, boron addend, toxin, immunomodulator, photoactive agent ordye, cancer chemotherapeutic drug, antiviral drug, antifungal drug,antibacterial drug, antiprotozoal drug and a chemosensitizing agent. 27.The method of claim 26, wherein said radioisotope is selected from thegroup consisting of α-emitters, β-emitters, Auger electron emitters,neutron capturing agents that emit α-particles and radioisotopes thatdecay by electron capture.
 28. The method of claim 26, wherein saidradioisotope is selected from the group consisting of ¹⁹⁸Au, ³²P, ¹²⁵I,¹³¹I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu and ²¹¹At.